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Abstract:

The present invention concerns methods and reagents useful in modulating
gene expression in a variety of applications, including use in
therapeutic, diagnostic, target validation, and genomic discovery
applications. Specifically, the invention relates to synthetic chemically
modified small nucleic acid molecules, such as short interfering nucleic
acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of
mediating RNA interference (RNAi) against target nucleic acid sequences.
The small nucleic acid molecules are useful in the treatment of any
disease or condition that responds to modulation of gene expression or
activity in a cell, tissue, or organism.

Claims:

1. A short interfering RNA (siRNA) molecule having a sense strand and an
antisense strand that mediates RNA interference, wherein: (a) each strand
is between 18 and 24 nucleotides in length; (b) the sense strand
comprises 10 or more 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, or
universal base modified nucleotides, and a terminal cap molecule at the
3'-end, the 5'-end, or both 3' and 5'-ends of the sense strand; (c) the
antisense strand comprises 10 or more 2'-deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, or universal base modified nucleotides; (d) 10 or
more pyrimidine nucleotides of the sense and/or antisense strand are
2'-deoxy, 2'-O-methyl or 2'-deoxy-2'-fluoro nucleotides; and (e) at least
one strand comprises a compound having Formula 119: ##STR00101##
wherein W comprises a linker molecule or chemical linkage that can be
present or absent, each R7 independently comprises an acetyl group, and
each n is independently an integer from 1 to 20.

2. The siRNA molecule of claim 1, wherein W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.

[0003] The following is a discussion of relevant art pertaining to RNAi.
The discussion is provided only for understanding of the invention that
follows. The summary is not an admission that any of the work described
below is prior art to the claimed invention. Applicant demonstrates
herein that chemically modified short interfering nucleic acids possess
the same capacity to mediate RNAi as do siRNA molecules and are expected
to possess improved stability and activity in vivo; therefore, this
discussion is not meant to be limiting only to siRNA and can be applied
to siNA as a whole.

[0004] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et
al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,
950-951). The corresponding process in plants is commonly referred to as
post-transcriptional gene silencing or RNA silencing and is also referred
to as quelling in fungi. The process of post-transcriptional gene
silencing is thought to be an evolutionarily-conserved cellular defense
mechanism used to prevent the expression of foreign genes and is commonly
shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15,
358). Such protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived from
viral infection or from the random integration of transposon elements
into a host genome via a cellular response that specifically destroys
homologous single-stranded RNA or viral genomic RNA. The presence of
dsRNA in cells triggers the RNAi response though a mechanism that has yet
to be fully characterized. This mechanism appears to be different from
the interferon response that results from dsRNA-mediated activation of
protein kinase PKR and 2',5'-oligoadenylate synthetase resulting in
non-specific cleavage of mRNA by ribonuclease L.

[0005] The presence of long dsRNAs in cells stimulates the activity of a
ribonuclease III enzyme referred to as dicer. Dicer is involved in the
processing of the dsRNA into short pieces of dsRNA known as short
interfering RNAs (siRNAs) (Hamilton et al., supra; Zamore et al., 2000,
Cell, 101, 25-33; Berstein et al., 2001, Nature, 409, 363). Short
interfering RNAs derived from dicer activity are typically about 21 to
about 23 nucleotides in length and comprise about 19 base pair duplexes
(Hamilton et al., supra; Elbashir et al., 2001, Genes Dev., 15, 188).
Dicer has also been implicated in the excision of 21- and 22-nucleotide
small temporal RNAs (stRNAs) from precursor RNA of conserved structure
that are implicated in translational control (Hutvagner et al., 2001,
Science, 293, 834). The RNAi response also features an endonuclease
complex, commonly referred to as an RNA-induced silencing complex (RISC),
which mediates cleavage of single-stranded RNA having sequence
complementary to the antisense strand of the siRNA duplex. Cleavage of
the target RNA takes place in the middle of the region complementary to
the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes
Dev., 15, 188).

[0006] RNAi has been studied in a variety of systems. Fire et al., 1998,
Nature, 391, 806, were the first to observe RNAi in C. elegans. Bahramian
and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny
and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by
dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293,
describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et
al., 2001, Nature, 411, 494, describe RNAi induced by introduction of
duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells
including human embryonic kidney and HeLa cells. Recent work in
Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877)
has revealed certain requirements for siRNA length, structure, chemical
composition, and sequence that are essential to mediate efficient RNAi
activity. These studies have shown that 21-nucleotide siRNA duplexes are
most active when containing 3'-terminal dinucleotide overhangs.
Furthermore, complete substitution of one or both siRNA strands with
2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi activity,
whereas substitution of the 3'-terminal siRNA overhang nucleotides with
2'-deoxy nucleotides (2'-H) was shown to be tolerated. Single mismatch
sequences in the center of the siRNA duplex were also shown to abolish
RNAi activity. In addition, these studies also indicate that the position
of the cleavage site in the target RNA is defined by the 5'-end of the
siRNA guide sequence rather than the 3'-end of the guide sequence
(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated
that a 5'-phosphate on the target-complementary strand of a siRNA duplex
is required for siRNA activity and that ATP is utilized to maintain the
5'-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

[0007] Studies have shown that replacing the 3'-terminal nucleotide
overhanging segments of a 21-mer siRNA duplex having two-nucleotide
3'-overhangs with deoxyribonucleotides does not have an adverse effect on
RNAi activity. Replacing up to four nucleotides on each end of the siRNA
with deoxyribonucleotides has been reported to be well tolerated, whereas
complete substitution with deoxyribonucleotides results in no RNAi
activity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition,
Elbashir et al., supra, also report that substitution of siRNA with
2'-O-methyl nucleotides completely abolishes RNAi activity. Li et al.,
International PCT Publication No. WO 00/44914, and Beach et al.,
International PCT Publication No. WO 01/68836 preliminarily suggest that
siRNA may include modifications to either the phosphate-sugar backbone or
the nucleoside to include at least one of a nitrogen or sulfur
heteroatom, however, neither application postulates to what extent such
modifications would be tolerated in siRNA molecules, nor provides any
further guidance or examples of such modified siRNA. Kreutzer et al.,
Canadian Patent Application No. 2,359,180, also describe certain chemical
modifications for use in dsRNA constructs in order to counteract
activation of double-stranded RNA-dependent protein kinase PKR,
specifically 2'-amino or 2'-β-methyl nucleotides, and nucleotides
containing a 2'-O or 4'-C methylene bridge. However, Kreutzer et al.
similarly fails to provide examples or guidance as to what extent these
modifications would be tolerated in siRNA molecules.

[0008] Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certain
chemical modifications targeting the unc-22 gene in C. elegans using long
(>25 nt) siRNA transcripts. The authors describe the introduction of
thiophosphate residues into these siRNA transcripts by incorporating
thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and
observed that RNAs with two phosphorothioate modified bases also had
substantial decreases in effectiveness as RNAi. Further, Parrish et al.
reported that phosphorothioate modification of more than two residues
greatly destabilized the RNAs in vitro such that interference activities
could not be assayed. Id. at 1081. The authors also tested certain
modifications at the 2'-position of the nucleotide sugar in the long
siRNA transcripts and found that substituting deoxynucleotides for
ribonucleotides produced a substantial decrease in interference activity,
especially in the case of Uridine to Thymidine and/or Cytidine to
deoxy-Cytidine substitutions. Id. In addition, the authors tested certain
base modifications, including substituting, in sense and antisense
strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and
3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas
4-thiouracil and 5-bromouracil substitution appeared to be tolerated,
Parrish reported that inosine produced a substantial decrease in
interference activity when incorporated in either strand. Parrish also
reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in
the antisense strand resulted in a substantial decrease in RNAi activity
as well.

[0011] This invention relates to compounds, compositions, and methods
useful for modulating RNA function and/or gene expression in a cell.
Specifically, the instant invention features synthetic small nucleic acid
molecules, such as short interfering nucleic acid (siNA), short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),
and short hairpin RNA (shRNA) molecules capable of modulating gene
expression in cells by RNA inference (RNAi). The siNA molecules of the
invention can be chemically modified. The use of chemically modified siNA
can improve various properties of native siRNA molecules through
increased resistance to nuclease degradation in vivo and/or improved
cellular uptake. The chemically modified siNA molecules of the instant
invention provide useful reagents and methods for a variety of
therapeutic, diagnostic, agricultural, target validation, genomic
discovery, genetic engineering and pharmacogenomic applications.

[0012] In a non-limiting example, the introduction of chemically modified
nucleotides into nucleic acid molecules provides a powerful tool in
overcoming potential limitations of in vivo stability and bioavailability
inherent to native RNA molecules that are delivered exogenously. For
example, the use of chemically modified nucleic acid molecules can enable
a lower dose of a particular nucleic acid molecule for a given
therapeutic effect since chemically modified nucleic acid molecules tend
to have a longer half-life in serum. Furthermore, certain chemical
modifications can improve the bioavailability of nucleic acid molecules
by targeting particular cells or tissues and/or improving cellular uptake
of the nucleic acid molecule. Therefore, even if the activity of a
chemically modified nucleic acid molecule is reduced as compared to a
native nucleic acid molecule, for example when compared to an all RNA
nucleic acid molecule, the overall activity of the modified nucleic acid
molecule can be greater than the native molecule due to improved
stability and/or delivery of the molecule. Unlike native unmodified
siRNA, chemically modified siNA can also minimize the possibility of
activating interferon activity in humans.

[0013] In one embodiment, the nucleic acid molecules of the invention that
act as mediators of the RNA interference gene silencing response are
chemically modified double stranded nucleic acid molecules. As in their
native double stranded RNA counterparts, these siNA molecules typically
consist of duplexes containing about 19 base pairs between
oligonucleotides comprising about 19 to about 25 nucleotides. The most
active siRNA molecules are thought to have such duplexes with overhanging
ends of 1-3 nucleotides, for example 21 nucleotide duplexes with 19 base
pairs and 2 nucleotide 3'-overhangs. These overhanging segments are
readily hydrolyzed by endonucleases in vivo. Studies have shown that
replacing the 3'-overhanging segments of a 21-mer siRNA duplex having 2
nucleotide 3' overhangs with deoxyribonucleotides does not have an
adverse effect on RNAi activity. Replacing up to 4 nucleotides on each
end of the siRNA with deoxyribonucleotides has been reported to be well
tolerated whereas complete substitution with deoxyribonucleotides results
in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). In
addition, Elbashir et al, supra, also report that substitution of siRNA
with 2'-O-methyl nucleotides completely abolishes RNAi activity. Li et
al., International PCT Publication No. WO 00/44914, and Beach et al.,
International PCT Publication No. WO 01/68836 both suggest that siRNA may
include modifications to either the phosphate-sugar back bone or the
nucleoside to include at least one of a nitrogen or sulfur heteroatom,
however neither application teaches to what extent these modifications
are tolerated in siRNA molecules nor provide any examples of such
modified siRNA. Kreutzer and Limmer, Canadian Patent Application No.
2,359,180, also describe certain chemical modifications for use in dsRNA
constructs in order to counteract activation of double
stranded-RNA-dependent protein kinase PKR, specifically 2'-amino or
2'-O-methyl nucleotides, and nucleotides containing a 2'-O or 4'-C
methylene bridge. However, Kreutzer and Limmer similarly fail to show to
what extent these modifications are tolerated in siRNA molecules nor
provide any examples of such modified siRNA.

[0014] In one embodiment, the invention features chemically modified siNA
constructs having specificity for target nucleic acid molecules in a
cell. Non-limiting examples of such chemical modifications include
without limitation phosphorothioate internucleotide linkages, 2'-O-methyl
ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy
ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides,
and inverted deoxyabasic residue incorporation. These chemical
modifications, when used in various siNA constructs, are shown to
preserve RNAi activity in cells while at the same time, dramatically
increasing the serum stability of these compounds. Furthermore, contrary
to the data published by Parrish et al., supra, applicant demonstrates
that multiple (greater than one) phosphorothioate substitutions are
well-tolerated and confer substantial increases in serum stability for
modified siNA constructs.

[0015] In one embodiment, the chemically-modified siNA molecules of the
invention comprise a duplex having two strands, one or both of which can
be chemically-modified, wherein each strand is about 19 to about 29
(e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) (e.g., about
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In one
embodiment, the chemically-modified siNA molecules of the invention
comprise a duplex having two strands, one or both of which can be
chemically-modified, wherein each strand is about 19 to about 23 (e.g.,
about 19, 20, 21, 22, or 23) nucleotides. In one embodiment, a siNA
molecule of the invention comprises modified nucleotides while
maintaining the ability to mediate RNAi. The modified nucleotides can be
used to improve in vitro or in vivo characteristics such as stability,
activity, and/or bioavailability. For example, a siNA molecule of the
invention can comprise modified nucleotides as a percentage of the total
number of nucleotides present in the siNA molecule. As such, a siNA
molecule of the invention can generally comprise modified nucleotides
from about 5 to about 100% of the nucleotide positions (e.g., 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 100% of the nucleotide positions). The actual percentage
of modified nucleotides present in a given siNA molecule depends on the
total number of nucleotides present in the siNA. If the siNA molecule is
single stranded, the percent modification can be based upon the total
number of nucleotides present in the single stranded siNA molecules.
Likewise, if the siNA molecule is double stranded, the percent
modification can be based upon the total number of nucleotides present in
the sense strand, antisense strand, or both the sense and antisense
strands. In addition, the actual percentage of modified nucleotides
present in a given siNA molecule can also depend on the total number of
purine and pyrimidine nucleotides present in the siNA, for example,
wherein all pyrimidine nucleotides and/or all purine nucleotides present
in the siNA molecule are modified.

[0016] The antisense region of a siNA molecule of the invention can
comprise a phosphorothioate internucleotide linkage at the 3'-end of said
antisense region. The antisense region can comprise about one to about
five phosphorothioate internucleotide linkages at the 5'-end of said
antisense region. The 3'-terminal nucleotide overhangs of a siNA molecule
of the invention can comprise ribonucleotides or deoxyribonucleotides
that are chemically-modified at a nucleic acid sugar, base, or backbone.
The 3'-terminal nucleotide overhangs can comprise one or more universal
base ribonucleotides. The 3'-terminal nucleotide overhangs can comprise
one or more acyclic nucleotides.

[0017] In one embodiment, a siNA molecule of the invention comprises blunt
ends, i.e., the ends do not include any overhanging nucleotides. For
example, a siNA molecule of the invention comprising modifications
described herein (e.g., comprising nucleotides having Formulae I-VII or
siNA constructs comprising Stab1-Stab18 or any combination thereof)
and/or any length described herein can comprise blunt ends or ends with
no overhanging nucleotides.

[0018] In one embodiment, any siNA molecule of the invention can comprise
one or more blunt ends, i.e. where a blunt end does not have any
overhanging nucleotides. In a non-limiting example, a blunt ended siNA
molecule has a number of base pairs equal to the number of nucleotides
present in each strand of the siNA molecule. In another example, a siNA
molecule comprises one blunt end, for example wherein the 5'-end of the
antisense strand and the 3'-end of the sense strand do not have any
overhanging nucleotides. In another example, a siNA molecule comprises
one blunt end, for example wherein the 3'-end of the antisense strand and
the 5'-end of the sense strand do not have any overhanging nucleotides.
In another example, a siNA molecule comprises two blunt ends, for example
wherein the 3'-end of the antisense strand and the 5'-end of the sense
strand as well as the 5'-end of the antisense strand and 3'-end of the
sense strand do not have any overhanging nucleotides. A blunt ended siNA
molecule can comprise, for example, from about 18 to about 30 nucleotides
(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides). Other nucleotides present in a blunt ended siNA molecule
can comprise mismatches, bulges, loops, or wobble base pairs, for
example, to modulate the activity of the siNA molecule to mediate RNA
interference.

[0019] By "blunt ends" is meant symmetric termini or termini of a double
stranded siNA molecule having no overhanging nucleotides. The two strands
of a double stranded siNA molecule align with each other without
over-hanging nucleotides at the termini. For example, a blunt ended siNA
construct comprises terminal nucleotides that are complimentary between
the sense and antisense regions of the siNA molecule.

[0020] In one embodiment, the invention features the use of a
double-stranded short interfering nucleic acid (siNA) molecule to
down-regulate expression of a target gene, wherein the siNA molecule
comprises one or more chemical modifications and each strand of the
double-stranded siNA is about 19 to about 23 nucleotides (e.g., about 19,
20, 21, 22, or 23 nucleotides) long.

[0021] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates expression
of a target gene, wherein the siNA molecule comprises no ribonucleotides
and each strand of the double-stranded siNA comprises about 19 to about
23 nucleotides.

[0022] In one embodiment, one of the strands of a double-stranded siNA
molecule of the invention comprises a nucleotide sequence that is
complementary to a nucleotide sequence or a portion thereof of a target
gene, and wherein the second strand of a double-stranded siNA molecule
comprises a nucleotide sequence substantially similar to the nucleotide
sequence or a portion thereof of the target gene.

[0023] In one embodiment, a siNA molecule of the invention comprises about
19 to about 23 nucleotides, and each strand comprises at least about 19
nucleotides that are complementary to the nucleotides of the other
strand.

[0024] In one embodiment, a siNA molecule of the invention comprises an
antisense region comprising a nucleotide sequence that is complementary
to a nucleotide sequence or a portion thereof of a target gene, and the
siNA further comprises a sense region, wherein the sense region comprises
a nucleotide sequence substantially similar to the nucleotide sequence or
a portion thereof of the target gene. The antisense region and the sense
region each comprise about 19 to about 23 nucleotides, and the antisense
region comprises at least about 19 nucleotides that are complementary to
nucleotides of the sense region.

[0025] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein the antisense region
comprises a nucleotide sequence that is complementary to a nucleotide
sequence or a portion thereof of RNA encoded by a target gene and the
sense region comprises a nucleotide sequence that is complementary to the
antisense region.

[0026] In one embodiment, a siNA molecule of the invention is assembled
from two separate oligonucleotide fragments wherein one fragment
comprises the sense region and the second fragment comprises the
antisense region of the siNA molecule. In another embodiment, the sense
region is connected to the antisense region via a linker molecule, which
can be a polynucleotide linker or a non-nucleotide linker.

[0027] In one embodiment, a siNA molecule of the invention comprises a
sense region and antisense region, wherein pyrimidine nucleotides in the
sense region comprises 2'-β-methylpyrimidine nucleotides and purine
nucleotides in the sense region comprise 2'-deoxy purine nucleotides. In
one embodiment, a siNA molecule of the invention comprises a sense region
and antisense region, wherein pyrimidine nucleotides present in the sense
region comprise 2'-deoxy-2'-fluoro pyrimidine nucleotides and wherein
purine nucleotides present in the sense region comprise 2'-deoxy purine
nucleotides.

[0028] In one embodiment, a siNA molecule of the invention comprises a
sense region and antisense region, wherein the pyrimidine nucleotides
when present in said antisense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides and the purine nucleotides when present in said antisense
region are 2'-O-methyl purine nucleotides.

[0029] In one embodiment, a siNA molecule of the invention comprises a
sense region and antisense region, wherein the pyrimidine nucleotides
when present in said antisense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides and wherein the purine nucleotides when present in said
antisense region comprise 2'-deoxy-purine nucleotides.

[0030] In one embodiment, a siNA molecule of the invention comprises a
sense region and antisense region, wherein the sense region includes a
terminal cap moiety at the 5'-end, the 3'-end, or both of the 5' and 3'
ends of the sense region. In another embodiment, the terminal cap moiety
is an inverted deoxy abasic moiety.

[0031] In one embodiment, a siNA molecule of the invention has RNAi
activity that modulates expression of RNA encoded by a gene. Because many
genes can share some degree of sequence homology with each other, siNA
molecules can be designed to target a class of genes (and associated
receptor or ligand genes) or alternately specific genes by selecting
sequences that are either shared amongst different gene targets or
alternatively that are unique for a specific gene target. Therefore, in
one embodiment, the siNA molecule can be designed to target conserved
regions of a RNA sequence having homology between several genes so as to
target several genes or gene families (e.g., different gene isoforms,
splice variants, mutant genes etc.) with one siNA molecule. In another
embodiment, the siNA molecule can be designed to target a sequence that
is unique to a specific RNA sequence of a specific gene due to the high
degree of specificity that the siNA molecule requires to mediate RNAi
activity.

[0032] In one embodiment, nucleic acid molecules of the invention that act
as mediators of the RNA interference gene silencing response are
double-stranded nucleic acid molecules. In another embodiment, the siNA
molecules of the invention consist of duplexes containing about 19 base
pairs between oligonucleotides comprising about 19 to about 25 (e.g.,
about 19, 20, 21, 22, 23, 24 or 25) nucleotides. In yet another
embodiment, siNA molecules of the invention comprise duplexes with
overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3)
nucleotides, for example, about 21-nucleotide duplexes with about 19 base
pairs and 3'-terminal mononucleotide, dinucleotide, or trinucleotide
overhangs.

[0033] In one embodiment, the invention features one or more
chemically-modified siNA constructs having specificity for nucleic acid
molecules that express or encode a protein sequence, such as RNA or DNA
encoding a protein sequence. Non-limiting examples of such chemical
modifications include without limitation phosphorothioate internucleotide
linkages, 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides,
2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides,
"acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl
and/or inverted deoxy abasic residue incorporation. These chemical
modifications, when used in various siNA constructs, are shown to
preserve RNAi activity in cells while at the same time, dramatically
increasing the serum stability of these compounds.

[0034] In one embodiment, a siNA molecule of the invention does not
contain any ribonucleotides. In another embodiment, a siNA molecule of
the invention comprises one or more ribonucleotides.

[0035] In one embodiment, the invention features the use of compounds or
compositions that inhibit the activity of double stranded RNA binding
proteins (dsRBPs, see for example Silhavy et al., 2003, Journal of
General Virology, 84, 975-980). Non-limiting examples of compounds and
compositions that can be used to inhibit the activity of dsRBPs include
but are not limited to small molecules and nucleic acid aptamers that
bind to or interact with the dsRBPs and consequently reduce dsRBP
activity and/or siNA molecules that target nucleic acid sequences
encoding dsRBPs. The use of such compounds and compositions is expected
to improve the activity of siNA molecules in biological systems in which
dsRBPs can abrogate or suppress the efficacy of siNA mediated RNA
interference, such as where dsRBPs are expressed during viral infection
of a cell to escape RNAi surveillance. Therefore, the use of agents that
inhibit dsRBP activity is preferred in those instances where RNA
interference activity can be improved via the abrogation or suppression
of dsRBP activity. Such anti-dsRBP agents can be administered alone or
can be co-administered with siNA molecules of the invention, or can be
used to pretreat cells or a subject before siNA administration. In
another embodiment, anti-dsRBP agents are used to treat viral infection,
such as HCV, HBV, or HIV infection with or without siNA molecules of the
invention.

[0036] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates expression
of a gene, wherein one of the strands of the double-stranded siNA
molecule comprises a nucleotide sequence that is complementary to a
nucleotide sequence of the gene or RNA encoded by the gene or a portion
thereof, and wherein the second strand of the double-stranded siNA
molecule comprises a nucleotide sequence substantially similar to the
nucleotide sequence of the gene or RNA encoded by the gene or a portion
thereof.

[0037] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates expression
of a gene, wherein each strand of the siNA molecule comprises about 19 to
about 23 nucleotides, and wherein each strand comprises at least about 19
nucleotides that are complementary to the nucleotides of the other
strand.

[0038] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates expression
of a gene, wherein the siNA molecule comprises an antisense region
comprising a nucleotide sequence that is complementary to a nucleotide
sequence of the gene or RNA encoded by the gene or a portion thereof, and
wherein the siNA further comprises a sense region, wherein the sense
region comprises a nucleotide sequence substantially similar to the
nucleotide sequence of the gene or RNA encoded by the gene or a portion
thereof.

[0039] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that inhibits the expression of
a target gene by mediating RNA interference (RNAi) process, wherein the
siNA molecule comprises no ribonucleotides and wherein each strand of the
double-stranded siNA molecule comprises about 21 nucleotides.

[0040] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that inhibits the replication of
a virus (e.g, as mammalian virus, plant virus, hepatitis C virus, human
immunodeficiency virus, hepatitis B virus, herpes simplex virus,
cytomegalovirus, human papilloma virus, respiratory syncytial virus, or
influenza virus), wherein the siNA molecule does not require the presence
of a ribonucleotide within the siNA molecule for the inhibition of
replication of the virus and each strand of the double-stranded siNA
molecule comprises about 21 nucleotides.

[0041] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates expression
of a gene, wherein the siNA molecule comprises a sense region and an
antisense region and wherein the antisense region comprises a nucleotide
sequence that is complementary to a nucleotide sequence or a portion
thereof of RNA encoded by the gene and the sense region comprises a
nucleotide sequence that is complementary to the antisense region or a
portion thereof, and wherein the purine nucleotides present in the
antisense region comprise 2'-deoxy-purine nucleotides. In another
embodiment, the purine nucleotides present in the antisense region
comprise 2'-O-methyl purine nucleotides. In either of the above
embodiments, the antisense region comprises a phosphorothioate
internucleotide linkage at the 3' end of the antisense region. In an
alternative embodiment, the antisense region comprises a glyceryl
modification at the 3' end of the antisense region. In another embodiment
of any of the above described siNA molecules, any nucleotides present in
a non-complementary region of the antisense strand (e.g. overhang region)
are 2'-deoxy nucleotides.

[0042] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that down-regulates expression
of a gene, wherein the siNA molecule is assembled from two separate
oligonucleotide fragments each comprising 21 nucleotides, wherein one
fragment comprises the sense region and the second fragment comprises the
antisense region of the siNA molecule, and wherein about 19 nucleotides
of each fragment of the siNA molecule are base-paired to the
complementary nucleotides of the other fragment of the siNA molecule and
wherein at least two 3' terminal nucleotides of each fragment of the siNA
molecule are not base-paired to the nucleotides of the other fragment of
the siNA molecule. In one embodiment, each of the two 3' terminal
nucleotides of each fragment of the siNA molecule is a
2'-deoxy-pyrimidine nucleotide, such as 2'-deoxy-thymidine. In another
embodiment, all 21 nucleotides of each fragment of the siNA molecule are
base-paired to the complementary nucleotides of the other fragment of the
siNA molecule. In another embodiment, about 19 nucleotides of the
antisense region are base-paired to the nucleotide sequence or a portion
thereof of the RNA encoded by the gene. In another embodiment, 21
nucleotides of the antisense region are base-paired to the nucleotide
sequence or a portion thereof of the RNA encoded by the gene. In any of
the above embodiments, the 5'-end of the fragment comprising said
antisense region can optionally include a phosphate group.

[0043] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that inhibits the expression of
a RNA sequence (e.g., wherein said target RNA sequence is encoded by a
gene or a gene involved in a pathway of gene expression), wherein the
siNA molecule does not contain any ribonucleotides and wherein each
strand of the double-stranded siNA molecule is about 21 nucleotides long.

[0044] In one embodiment, the invention features a medicament comprising a
siNA molecule of the invention.

[0045] In one embodiment, the invention features an active ingredient
comprising a siNA molecule of the invention.

[0046] In one embodiment, the invention features the use of a
double-stranded short interfering nucleic acid (siNA) molecule to
down-regulate expression of a target gene, wherein the siNA molecule
comprises one or more chemical modifications and each strand of the
double-stranded siNA is about 21 nucleotides long.

[0047] The invention features a double-stranded short interfering nucleic
acid (siNA) molecule that inhibits expression of a gene, wherein one of
the strands of the double-stranded siNA molecule is an antisense strand
which comprises nucleotide sequence that is complementary to nucleotide
sequence of a RNA encoded by the gene or a portion thereof, the other
strand is a sense strand which comprises nucleotide sequence that is
complementary to a nucleotide sequence of the antisense strand and
wherein a majority of the pyrimidine nucleotides present in the
double-stranded siNA molecule comprises a sugar modification. In one
embodiment, the nucleotide sequence of the antisense strand of the
double-stranded siNA molecule is complementary to the nucleotide sequence
of a RNA which encodes a protein or a portion thereof. In one embodiment,
each strand of the siNA molecule comprises about 19 to about 29 (e.g.,
about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, and
each strand comprises at least about 19 nucleotides that are
complementary to the nucleotides of the other strand. In one embodiment,
the siNA molecule is assembled from two oligonucleotide fragments,
wherein one fragment comprises the nucleotide sequence of the antisense
strand of the siNA molecule and a second fragment comprises nucleotide
sequence of the sense region of the siNA molecule. In another embodiment,
the sense strand is connected to the antisense strand via a linker
molecule, such as a polynucleotide linker or a non-nucleotide linker. In
one embodiment, the pyrimidine nucleotides present in the sense strand
are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides
present in the sense region are 2'-deoxy purine nucleotides. In another
embodiment, the pyrimidine nucleotides present in the sense strand are
2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine nucleotides
present in the sense region are 2'-O-methyl purine nucleotides. In one
embodiment, the sense strand comprises a 3'-end and a 5'-end, wherein a
terminal cap moiety (e.g., an inverted deoxy abasic moiety) is present at
the 5'-end, the 3'-end, or both of the 5' and 3' ends of the sense
strand. In one embodiment, the antisense strand comprises one or more
2'-deoxy-2'-fluoro pyrimidine nucleotides and one or more 2'-O-methyl
purine nucleotides. In one embodiment, the pyrimidine nucleotides present
in the antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and
any purine nucleotides present in the antisense strand are 2'-O-methyl
purine nucleotides. In one embodiment, the antisense strand comprises a
phosphorothioate internucleotide linkage at the 3' end of the antisense
strand. In another embodiment, the antisense strand comprises a glyceryl
modification at the 3' end. In another embodiment, the 5'-end of the
antisense strand optionally includes a phosphate group. In one
embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a gene,
wherein one of the strands of the double-stranded siNA molecule is an
antisense strand which comprises nucleotide sequence that is
complementary to nucleotide sequence of RNA encoded by a gene or a
portion thereof, the other strand is a sense strand which comprises
nucleotide sequence that is complementary to a nucleotide sequence of the
antisense strand and wherein a majority of the pyrimidine nucleotides
present in the double-stranded siNA molecule comprises a sugar
modification, and wherein the nucleotide sequence of the antisense strand
is complementary to a nucleotide sequence of the 5'-untranslated region
or a portion thereof of the RNA. In another embodiment, the nucleotide
sequence of the antisense strand is complementary to a nucleotide
sequence of the RNA or a portion thereof.

[0048] In one embodiment, the invention features a double-stranded short
interfering nucleic acid (siNA) molecule that inhibits expression of a
gene, wherein one of the strands of the double-stranded siNA molecule is
an antisense strand which comprises nucleotide sequence that is
complementary to nucleotide sequence of a RNA or a portion thereof, the
other strand is a sense strand which comprises nucleotide sequence that
is complementary to a nucleotide sequence of the antisense strand and
wherein a majority of the pyrimidine nucleotides present in the
double-stranded siNA molecule comprises a sugar modification, and wherein
each of the two strands of the siNA molecule comprises 21 nucleotides. In
one embodiment, about 19 nucleotides of each strand of the siNA molecule
are base-paired to the complementary nucleotides of the other strand of
the siNA molecule and at least two 3' terminal nucleotides of each strand
of the siNA molecule are not base-paired to the nucleotides of the other
strand of the siNA molecule.

[0049] In one embodiment, each of the two 3' terminal nucleotides of each
fragment of the siNA molecule are 2'-deoxy-pyrimidines, such as
2'-deoxy-thymidine. In another embodiment, each strand of the siNA
molecule is base-paired to the complementary nucleotides of the other
strand of the siNA molecule. In one embodiment, about 19 nucleotides of
the antisense strand are base-paired to the nucleotide sequence of the
RNA or a portion thereof. In another embodiment, 21 nucleotides of the
antisense strand are base-paired to the nucleotide sequence of the RNA or
a portion thereof.

[0050] In one embodiment, the invention features a composition comprising
a siNA molecule of the invention and a pharmaceutically acceptable
carrier or diluent.

[0051] In one embodiment, the invention features a method of increasing
the stability of a siNA molecule against cleavage by ribonucleases
comprising introducing at least one modified nucleotide into the siNA
molecule, wherein the modified nucleotide is a 2'-deoxy-2'-fluoro
nucleotide. In another embodiment, all pyrimidine nucleotides present in
the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In another
embodiment, the modified nucleotides in the siNA include at least one
2'-deoxy-2'-fluoro cytidine or 2'-deoxy-2'-fluoro uridine nucleotide. In
another embodiment, the modified nucleotides in the siNA include at least
one 2'-fluoro cytidine and at least one 2'-deoxy-2'-fluoro uridine
nucleotides. In another embodiment, all uridine nucleotides present in
the siNA are 2'-deoxy-2'-fluoro uridine nucleotides. In another
embodiment, all cytidine nucleotides present in the siNA are
2'-deoxy-2'-fluoro cytidine nucleotides. In another embodiment, all
adenosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
adenosine nucleotides. In another embodiment, all guanosine nucleotides
present in the siNA are 2'-deoxy-2'-fluoro guanosine nucleotides. The
siNA can further comprise at least one modified internucleotidic linkage,
such as phosphorothioate linkage. In another embodiment, the
2'-deoxy-2'-fluoronucleotides are present at specifically selected
locations in the siNA that are sensitive to cleavage by ribonucleases,
such as locations having pyrimidine nucleotides.

[0052] In one embodiment, the invention features the use of a
double-stranded short interfering nucleic acid (siNA) molecule that
inhibits expression of a gene, wherein one of the strands of the
double-stranded siNA molecule is an antisense strand which comprises
nucleotide sequence that is complementary to nucleotide sequence of a RNA
or a portion thereof, the other strand is a sense strand which comprises
nucleotide sequence that is complementary to a nucleotide sequence of the
antisense strand and wherein a majority of the pyrimidine nucleotides
present in the double-stranded siNA molecule comprises a sugar
modification.

[0053] In one embodiment, the invention features a short interfering
nucleic acid (siNA) molecule comprising a double-stranded structure that
down-regulates expression of a target nucleic acid, wherein the siNA
molecule does not require a 2'-hydroxyl group containing ribonucleotide,
each strand of the double-stranded structure of the siNA molecule
comprises about 21 nucleotides and the siNA molecule comprises nucleotide
sequence having complementarity to nucleotide sequence of the target
nucleic acid or a portion thereof. The target nucleic acid can be an
endogenous gene, an exogenous gene, a viral nucleic acid, or a RNA, such
as a mammalian gene, plant gene, viral gene, fungal gene, bacterial gene,
plant viral gene, or mammalian viral gene. Examples of mammalian viral
gene include hepatitis C virus, human immunodeficiency virus, hepatitis B
virus, herpes simplex virus, cytomegalovirus, human papilloma virus,
respiratory syncytial virus, influenza virus, and severe acute
respiratory syndrome virus (SARS).

[0054] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region wherein the antisense region
comprises the nucleotide sequence that is complementary to a nucleotide
sequence or a portion thereof of the target nucleic acid and the sense
region comprises a nucleotide sequence that is complementary to
nucleotide sequence of the antisense region or a portion thereof.

[0055] In one embodiment, a siNA molecule of the invention is assembled
from two separate oligonucleotide fragments wherein one fragment
comprises the sense region and the second fragment comprises the
antisense region of the siNA molecule. The sense region can be connected
to the antisense region via a linker molecule, such as a polynucleotide
linker or non-nucleotide linker. In another embodiment, each sense region
and antisense region comprise about 21 nucleotides in length. In another
embodiment, about 19 nucleotides of each fragment of the siNA molecule
are base-paired to the complementary nucleotides of the other fragment of
the siNA molecule and at least two 3' terminal nucleotides of each
fragment of the siNA molecule are not base-paired to the nucleotides of
the other fragment of the siNA molecule. In another embodiment, each of
the two 3' terminal nucleotides of each fragment of the siNA molecule are
2'-deoxy-pyrimidines, such as the thymidine. In another embodiment, all
21 nucleotides of each fragment of the siNA molecule are base-paired to
the complementary nucleotides of the other fragment of the siNA molecule.
In another embodiment, about 19 nucleotides of the antisense region of
the siNA molecule are base-paired to the nucleotide sequence or a portion
thereof of the target nucleic acid. In another embodiment, 21 nucleotides
of the antisense region of the siNA molecule are base-paired to the
nucleotide sequence or a portion thereof of the target nucleic acid. In
another embodiment, the 5'-end of the fragment comprising the antisense
region optionally includes a phosphate group.

[0056] In one embodiment, a siNA molecule of the invention comprises
nucleotide sequence having complementarity to nucleotide sequence of RNA
or a portion thereof encoded by the target nucleic acid or a portion
thereof.

[0057] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein the pyrimidine nucleotides
when present in the sense region are 2'-O-methylpyrimidine nucleotides
and wherein the purine nucleotides when present in the sense region are
2'-deoxy purine nucleotides.

[0058] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein the pyrimidine nucleotides
when present in the sense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides and wherein the purine nucleotides when present in the sense
region are 2'-deoxy purine nucleotides.

[0059] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein the sense region includes a
terminal cap moiety at the 5'-end, the 3'-end, or both of the 5' and 3'
ends. The cap moiety can be an inverted deoxy abasic moiety, an inverted
deoxy thymidine moiety, or a thymidine moiety.

[0060] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein the pyrimidine nucleotides
when present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides and the purine nucleotides when present in the antisense
region are 2'-O-methyl purine nucleotides.

[0061] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein the pyrimidine nucleotides
when present in the antisense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides and wherein the purine nucleotides when present in the
antisense region comprise 2'-deoxy-purine nucleotides.

[0062] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein the antisense region
comprises a phosphate backbone modification at the 3' end of the
antisense region. The phosphate backbone modification can be a
phosphorothioate.

[0063] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein the antisense region
comprises a glyceryl modification at the 3' end of the antisense region.

[0064] In one embodiment, a siNA molecule of the invention comprises a
sense region and an antisense region, wherein each of sense and the
antisense regions of the siNA molecule comprise about 21 nucleotides.

[0065] In a non-limiting example, the introduction of chemically-modified
nucleotides into nucleic acid molecules provides a powerful tool in
overcoming potential limitations of in vivo stability and bioavailability
inherent to native RNA molecules that are delivered exogenously. For
example, the use of chemically-modified nucleic acid molecules can enable
a lower dose of a particular nucleic acid molecule for a given
therapeutic effect since chemically-modified nucleic acid molecules tend
to have a longer half-life in serum. Furthermore, certain chemical
modifications can improve the bioavailability of nucleic acid molecules
by targeting particular cells or tissues and/or improving cellular uptake
of the nucleic acid molecule. Therefore, even if the activity of a
chemically-modified nucleic acid molecule is reduced as compared to a
native nucleic acid molecule, for example, when compared to an all-RNA
nucleic acid molecule, the overall activity of the modified nucleic acid
molecule can be greater than that of the native molecule due to improved
stability and/or delivery of the molecule. Unlike native unmodified siNA,
chemically-modified siNA can also minimize the possibility of activating
interferon activity in humans.

[0066] In any of the embodiments of siNA molecules described herein, the
antisense region of a siNA molecule of the invention can comprise a
phosphorothioate internucleotide linkage at the 3'-end of said antisense
region. In any of the embodiments of siNA molecules described herein, the
antisense region can comprise about one to about five phosphorothioate
internucleotide linkages at the 5'-end of said antisense region. In any
of the embodiments of siNA molecules described herein, the 3'-terminal
nucleotide overhangs of a siNA molecule of the invention can comprise
ribonucleotides or deoxyribonucleotides that are chemically-modified at a
nucleic acid sugar, base, or backbone. In any of the embodiments of siNA
molecules described herein, the 3'-terminal nucleotide overhangs can
comprise one or more universal base ribonucleotides. In any of the
embodiments of siNA molecules described herein, the 3'-terminal
nucleotide overhangs can comprise one or more acyclic nucleotides.

[0067] One embodiment of the invention provides an expression vector
comprising a nucleic acid sequence encoding at least one siNA molecule of
the invention in a manner that allows expression of the nucleic acid
molecule. Another embodiment of the invention provides a mammalian cell
comprising such an expression vector. The mammalian cell can be a human
cell. The siNA molecule of the expression vector can comprise a sense
region and an antisense region. The antisense region can comprise
sequence complementary to an RNA or DNA sequence encoding a protein or
polypeptide and the sense region can comprise sequence complementary to
the antisense region. The siNA molecule can comprise two distinct strands
having complementary sense and antisense regions. The siNA molecule can
comprise a single strand having complementary sense and antisense
regions.

[0069] wherein each R1 and R2 is independently any nucleotide,
non-nucleotide, or polynucleotide which can be naturally-occurring or
chemically-modified, each X and Y is independently O, S, N, alkyl, or
substituted alkyl, each Z and W is independently O, S, N, alkyl,
substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W,
X, Y, and Z are optionally not all O. In another embodiment, a backbone
modification of the invention comprises a phosphonoacetate and/or
thiophosphonoacetate internucleotide linkage (see for example Sheehan et
al., 2003, Nucleic Acids Research, 31, 4109-4118).

[0070] The chemically-modified internucleotide linkages having Formula I,
for example, wherein any Z, W, X, and/or Y independently comprises a
sulphur atom, can be present in one or both oligonucleotide strands of
the siNA duplex, for example, in the sense strand, the antisense strand,
or both strands. The siNA molecules of the invention can comprise one or
more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
chemically-modified internucleotide linkages having Formula I at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand,
the antisense strand, or both strands. For example, an exemplary siNA
molecule of the invention can comprise about 1 to about 5 or more (e.g.,
about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide
linkages having Formula I at the 5'-end of the sense strand, the
antisense strand, or both strands. In another non-limiting example, an
exemplary siNA molecule of the invention can comprise one or more (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with
chemically-modified internucleotide linkages having Formula I in the
sense strand, the antisense strand, or both strands. In yet another
non-limiting example, an exemplary siNA molecule of the invention can
comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
purine nucleotides with chemically-modified internucleotide linkages
having Formula I in the sense strand, the antisense strand, or both
strands. In another embodiment, a siNA molecule of the invention having
internucleotide linkage(s) of Formula I also comprises a
chemically-modified nucleotide or non-nucleotide having any of Formulae
I-VII.

[0071] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule capable of mediating RNA
interference (RNAi) inside a cell or reconstituted in vitro system,
wherein the chemical modification comprises one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides
having Formula II:

[0072] The chemically-modified nucleotide or non-nucleotide of Formula II
can be present in one or both oligonucleotide strands of the siNA duplex,
for example in the sense strand, the antisense strand, or both strands.
The siNA molecules of the invention can comprise one or more
chemically-modified nucleotide or non-nucleotide of Formula II at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand,
the antisense strand, or both strands. For example, an exemplary siNA
molecule of the invention can comprise about 1 to about 5 or more (e.g.,
about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or
non-nucleotides of Formula II at the 5'-end of the sense strand, the
antisense strand, or both strands. In anther non-limiting example, an
exemplary siNA molecule of the invention can comprise about 1 to about 5
or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified
nucleotides or non-nucleotides of Formula II at the 3'-end of the sense
strand, the antisense strand, or both strands.

[0073] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule capable of mediating RNA
interference (RNAi) inside a cell or reconstituted in vitro system,
wherein the chemical modification comprises one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides
having Formula III:

[0074] The chemically-modified nucleotide or non-nucleotide of Formula III
can be present in one or both oligonucleotide strands of the siNA duplex,
for example, in the sense strand, the antisense strand, or both strands.
The siNA molecules of the invention can comprise one or more
chemically-modified nucleotide or non-nucleotide of Formula III at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense strand,
the antisense strand, or both strands. For example, an exemplary siNA
molecule of the invention can comprise about 1 to about 5 or more (e.g.,
about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or
non-nucleotide(s) of Formula III at the 5'-end of the sense strand, the
antisense strand, or both strands. In another non-limiting example, an
exemplary siNA molecule of the invention can comprise about 1 to about 5
or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified
nucleotide or non-nucleotide of Formula III at the 3'-end of the sense
strand, the antisense strand, or both strands.

[0075] In another embodiment, a siNA molecule of the invention comprises a
nucleotide having Formula II or III, wherein the nucleotide having
Formula II or III is in an inverted configuration. For example, the
nucleotide having Formula II or III is connected to the siNA construct in
a 3'-3', 3'-2',2'-3', or 5'-5' configuration, such as at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of one or both siNA strands.

[0076] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule capable of mediating RNA
interference (RNAi) inside a cell or reconstituted in vitro system,
wherein the chemical modification comprises a 5'-terminal phosphate group
having Formula IV:

[0077] In one embodiment, the invention features a siNA molecule having a
5'-terminal phosphate group having Formula IV on the target-complementary
strand, for example, a strand complementary to a target RNA, wherein the
siNA molecule comprises an all RNA siNA molecule. In another embodiment,
the invention features a siNA molecule having a 5'-terminal phosphate
group having Formula IV on the target-complementary strand wherein the
siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3)
nucleotide 3'-terminal nucleotide overhangs having about 1 to about 4
(e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3'-end of one or
both strands. In another embodiment, a 5'-terminal phosphate group having
Formula IV is present on the target-complementary strand of a siNA
molecule of the invention, for example a siNA molecule having chemical
modifications having any of Formulae I-VII.

[0078] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule capable of mediating RNA
interference (RNAi) inside a cell or reconstituted in vitro system,
wherein the chemical modification comprises one or more phosphorothioate,
phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages.
For example, in a non-limiting example, the invention features a
chemically-modified short interfering nucleic acid (siNA) having about 1,
2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in
one siNA strand. In yet another embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) individually
having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate
internucleotide linkages in both siNA strands. The phosphorothioate
internucleotide linkages can be present in one or both oligonucleotide
strands of the siNA duplex, for example in the sense strand, the
antisense strand, or both strands. The siNA molecules of the invention
can comprise one or more phosphorothioate, phosphonoacetate, and/or
thiophosphonoacetate internucleotide linkages at the 3'-end, the 5'-end,
or both of the 3'- and 5'-ends of the sense strand, the antisense strand,
or both strands. For example, an exemplary siNA molecule of the invention
can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or
more) consecutive phosphorothioate internucleotide linkages at the 5'-end
of the sense strand, the antisense strand, or both strands. In another
non-limiting example, an exemplary siNA molecule of the invention can
comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
pyrimidine phosphorothioate internucleotide linkages in the sense strand,
the antisense strand, or both strands. In yet another non-limiting
example, an exemplary siNA molecule of the invention can comprise one or
more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine
phosphorothioate internucleotide linkages in the sense strand, the
antisense strand, or both strands.

[0079] In one embodiment, the invention features a siNA molecule, wherein
the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or
one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy,
2'-O-methyl, 2'-deoxy-2'-fluoro, and/or about one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides,
and optionally a terminal cap molecule at the 3'-end, the 5'-end, or both
of the 3'- and 5'-ends of the sense strand; and wherein the antisense
strand comprises about 1 to about 10 or more, specifically about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,
and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)
2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified
nucleotides, and optionally a terminal cap molecule at the 3'-end, the
5'-end, or both of the 3'- and 5'-ends of the antisense strand. In
another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense
siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl and/or
2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate
internucleotide linkages and/or a terminal cap molecule at the 3'-end,
the 5'-end, or both of the 3'- and 5'-ends, being present in the same or
different strand.

[0080] In another embodiment, the invention features a siNA molecule,
wherein the sense strand comprises about 1 to about 5, specifically about
1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or
more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or
more) universal base modified nucleotides, and optionally a terminal cap
molecule at the 3-end, the 5'-end, or both of the 3'- and 5'-ends of the
sense strand; and wherein the antisense strand comprises about 1 to about
5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or
one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal
base modified nucleotides, and optionally a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense
strand. In another embodiment, one or more, for example about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or
antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl
and/or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 5
or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate
internucleotide linkages and/or a terminal cap molecule at the 3'-end,
the 5'-end, or both of the 3'- and 5'-ends, being present in the same or
different strand.

[0081] In one embodiment, the invention features a siNA molecule, wherein
the antisense strand comprises one or more, for example, about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,
and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base
modified nucleotides, and optionally a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand;
and wherein the antisense strand comprises about 1 to about 10 or more,
specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or
one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal
base modified nucleotides, and optionally a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense
strand. In another embodiment, one or more, for example about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or
antisense siNA strand are chemically-modified with 2'-deoxy, 2'-O-methyl
and/or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for
example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate
internucleotide linkages and/or a terminal cap molecule at the 3'-end,
the 5'-end, or both of the 3' and 5'-ends, being present in the same or
different strand.

[0082] In another embodiment, the invention features a siNA molecule,
wherein the antisense strand comprises about 1 to about 5 or more,
specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide
linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base
modified nucleotides, and optionally a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand;
and wherein the antisense strand comprises about 1 to about 5 or more,
specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide
linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base
modified nucleotides, and optionally a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense
strand. In another embodiment, one or more, for example about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or
antisense siNA strand are chemically-modified with 2'-deoxy,
2'-β-methyl and/or 2'-deoxy-2'-fluoro nucleotides, with or without
about 1 to about 5, for example about 1, 2, 3, 4, 5 or more
phosphorothioate internucleotide linkages and/or a terminal cap molecule
at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends, being present
in the same or different strand.

[0083] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule having about 1 to about 5,
specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide
linkages in each strand of the siNA molecule.

[0084] In another embodiment, the invention features a siNA molecule
comprising 2'-5' internucleotide linkages. The 2'-5' internucleotide
linkage(s) can be at the 3'-end, the 5'-end, or both of the 3'- and
5'-ends of one or both siNA sequence strands. In addition, the 2'-5'
internucleotide linkage(s) can be present at various other positions
within one or both siNA sequence strands, for example, about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a
pyrimidine nucleotide in one or both strands of the siNA molecule can
comprise a 2'-5' internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more including every internucleotide linkage of a purine
nucleotide in one or both strands of the siNA molecule can comprise a
2'-5' internucleotide linkage.

[0085] In another embodiment, a chemically-modified siNA molecule of the
invention comprises a duplex having two strands, one or both of which can
be chemically-modified, wherein each strand is about 18 to about 27
(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in
length, wherein the duplex has about 18 to about 23 (e.g., about 18, 19,
20, 21, 22, or 23) base pairs, and wherein the chemical modification
comprises a structure having any of Formulae I-VII. For example, an
exemplary chemically-modified siNA molecule of the invention comprises a
duplex having two strands, one or both of which can be
chemically-modified with a chemical modification having any of Formulae
I-VII or any combination thereof, wherein each strand consists of about
21 nucleotides, each having a 2-nucleotide 3'-terminal nucleotide
overhang, and wherein the duplex has about 19 base pairs. In another
embodiment, a siNA molecule of the invention comprises a single stranded
hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about
36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18
to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and
wherein the siNA can include a chemical modification comprising a
structure having any of Formulae I-VII or any combination thereof. For
example, an exemplary chemically-modified siNA molecule of the invention
comprises a linear oligonucleotide having about 42 to about 50 (e.g.,
about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is
chemically-modified with a chemical modification having any of Formulae
I-VII or any combination thereof, wherein the linear oligonucleotide
forms a hairpin structure having about 19 base pairs and a 2-nucleotide
3'-terminal nucleotide overhang. In another embodiment, a linear hairpin
siNA molecule of the invention contains a stem loop motif, wherein the
loop portion of the siNA molecule is biodegradable. For example, a linear
hairpin siNA molecule of the invention is designed such that degradation
of the loop portion of the siNA molecule in vivo can generate a
double-stranded siNA molecule with 3'-terminal overhangs, such as
3'-terminal nucleotide overhangs comprising about 2 nucleotides.

[0086] In another embodiment, a siNA molecule of the invention comprises a
hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3
to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA
can include one or more chemical modifications comprising a structure
having any of Formulae I-VII or any combination thereof. For example, an
exemplary chemically-modified siNA molecule of the invention comprises a
linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is
chemically-modified with one or more chemical modifications having any of
Formulae I-VII or any combination thereof, wherein the linear
oligonucleotide forms a hairpin structure having about 3 to about 23
(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, or 23) base pairs and a 5'-terminal phosphate group that can
be chemically modified as described herein (for example a 5'-terminal
phosphate group having Formula IV). In another embodiment, a linear
hairpin siNA molecule of the invention contains a stem loop motif,
wherein the loop portion of the siNA molecule is biodegradable. In
another embodiment, a linear hairpin siNA molecule of the invention
comprises a loop portion comprising a non-nucleotide linker.

[0087] In another embodiment, a siNA molecule of the invention comprises
an asymmetric hairpin structure, wherein the siNA is about 25 to about 50
(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length
having about 3 to about 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20) base pairs, and wherein the siNA can
include one or more chemical modifications comprising a structure having
any of Formulae I-VII or any combination thereof. For example, an
exemplary chemically-modified siNA molecule of the invention comprises a
linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is
chemically-modified with one or more chemical modifications having any of
Formulae I-VII or any combination thereof, wherein the linear
oligonucleotide forms an asymmetric hairpin structure having about 3 to
about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17
or 18) base pairs and a 5'-terminal phosphate group that can be
chemically modified as described herein (for example a 5'-terminal
phosphate group having Formula IV). In another embodiment, an asymmetric
hairpin siNA molecule of the invention contains a stem loop motif,
wherein the loop portion of the siNA molecule is biodegradable. In
another embodiment, an asymmetric hairpin siNA molecule of the invention
comprises a loop portion comprising a non-nucleotide linker.

[0088] In another embodiment, a siNA molecule of the invention comprises
an asymmetric double stranded structure having separate polynucleotide
strands comprising sense and antisense regions, wherein the antisense
region is about 16 to about 25 (e.g., about 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25) nucleotides in length, wherein the sense region is about 3
to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, or 18) nucleotides in length, wherein the sense region the antisense
region have at least 3 complementary nucleotides, and wherein the siNA
can include one or more chemical modifications comprising a structure
having any of Formulae I-VII or any combination thereof. For example, an
exemplary chemically-modified siNA molecule of the invention comprises an
asymmetric double stranded structure having separate polynucleotide
strands comprising sense and antisense regions, wherein the antisense
region is about 18 to about 22 (e.g., about 18, 19, 20, 21, or 22)
nucleotides in length and wherein the sense region is about 3 to about 15
(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides
in length, wherein the sense region the antisense region have at least 3
complementary nucleotides, and wherein the siNA can include one or more
chemical modifications comprising a structure having any of Formulae
I-VII or any combination thereof. In another embodiment, the asymmetic
double stranded siNA molecule can also have a 5'-terminal phosphate group
that can be chemically modified as described herein (for example a
5'-terminal phosphate group having Formula IV).

[0089] In another embodiment, a siNA molecule of the invention comprises a
circular nucleic acid molecule, wherein the siNA is about 38 to about 70
(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length
having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base
pairs, and wherein the siNA can include a chemical modification, which
comprises a structure having any of Formulae I-VII or any combination
thereof. For example, an exemplary chemically-modified siNA molecule of
the invention comprises a circular oligonucleotide having about 42 to
about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides
that is chemically-modified with a chemical modification having any of
Formulae I-VII or any combination thereof, wherein the circular
oligonucleotide forms a dumbbell shaped structure having about 19 base
pairs and 2 loops.

[0090] In another embodiment, a circular siNA molecule of the invention
contains two loop motifs, wherein one or both loop portions of the siNA
molecule is biodegradable. For example, a circular siNA molecule of the
invention is designed such that degradation of the loop portions of the
siNA molecule in vivo can generate a double-stranded siNA molecule with
3'-terminal overhangs, such as 3'-terminal nucleotide overhangs
comprising about 2 nucleotides.

[0091] In one embodiment, a siNA molecule of the invention comprises at
least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic
moiety, for example a compound having Formula V:

[0094] In another embodiment, the invention features a compound having
Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3
comprises 0 and is the point of attachment to the 3'-end, the 5'-end, or
both of the 3' and 5'-ends of one or both strands of a double-stranded
siNA molecule of the invention or to a single-stranded siNA molecule of
the invention. This modification is referred to herein as "glyceryl" (for
example modification 6 in FIG. 22).

[0095] In another embodiment, a moiety having any of Formula V, VI or VII
of the invention is at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of a siNA molecule of the invention. For example, a moiety having
Formula V, VI or VII can be present at the 3'-end, the 5'-end, or both of
the 3' and 5'-ends of the antisense strand, the sense strand, or both
antisense and sense strands of the siNA molecule. In addition, a moiety
having Formula VII can be present at the 3'-end or the 5'-end of a
hairpin siNA molecule as described herein.

[0096] In another embodiment, a siNA molecule of the invention comprises
an abasic residue having Formula V or VI, wherein the abasic residue
having Formula V or VI is connected to the siNA construct in a 3-3',
3-2', 2-3', or 5-5' configuration, such as at the 3'-end, the 5'-end, or
both of the 3' and 5'-ends of one or both siNA strands.

[0097] In one embodiment, a siNA molecule of the invention comprises one
or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked
nucleic acid (LNA) nucleotides, for example at the 5'-end, the 3'-end,
both of the 5' and 3'-ends, or any combination thereof, of the siNA
molecule.

[0098] In another embodiment, a siNA molecule of the invention comprises
one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic
nucleotides, for example at the 5'-end, the 3'-end, both of the 5' and
3'-ends, or any combination thereof, of the siNA molecule.

[0099] In one embodiment, the sense strand of a double stranded siNA
molecule of the invention comprises a terminal cap moiety, (see for
example FIG. 22) such as an inverted deoxyabasic moiety or inverted
nucleotide, at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense
strand.

[0100] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention, wherein
the chemically-modified siNA comprises a sense region, where any (e.g.,
one or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all) purine
nucleotides present in the sense region are 2'-deoxy purine nucleotides
(e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or
alternately a plurality of purine nucleotides are 2'-deoxy purine
nucleotides).

[0101] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention, wherein
the chemically-modified siNA comprises a sense region, where any (e.g.,
one or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all) purine
nucleotides present in the sense region are 2'-deoxy purine nucleotides
(e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or
alternately a plurality of purine nucleotides are 2'-deoxy purine
nucleotides), wherein any nucleotides comprising a 3'-terminal nucleotide
overhang that are present in said sense region are 2'-deoxy nucleotides.

[0102] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention, wherein
the chemically-modified siNA comprises a sense region, where any (e.g.,
one or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all) purine
nucleotides present in the sense region are 2'-O-methyl purine
nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl purine nucleotides).

[0103] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention, wherein
the chemically-modified siNA comprises a sense region, where any (e.g.,
one or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where any (e.g., one or more or all) purine
nucleotides present in the sense region are 2'-O-methyl purine
nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl purine nucleotides), wherein any nucleotides comprising a
3'-terminal nucleotide overhang that are present in said sense region are
2'-deoxy nucleotides.

[0104] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention, wherein
the chemically-modified siNA comprises an antisense region, where any
(e.g., one or more or all) pyrimidine nucleotides present in the
antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides or alternately a plurality of pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., one or
more or all) purine nucleotides present in the antisense region are
2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-O-methyl purine nucleotides).

[0105] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention, wherein
the chemically-modified siNA comprises an antisense region, where any
(e.g., one or more or all) pyrimidine nucleotides present in the
antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides or alternately a plurality of pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., one or
more or all) purine nucleotides present in the antisense region are
2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-O-methyl purine nucleotides), wherein any nucleotides
comprising a 3'-terminal nucleotide overhang that are present in said
antisense region are 2'-deoxy nucleotides.

[0106] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention, wherein
the chemically-modified siNA comprises an antisense region, where any
(e.g., one or more or all) pyrimidine nucleotides present in the
antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides or alternately a plurality of pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and where any (e.g., one or
more or all) purine nucleotides present in the antisense region are
2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are
2'-deoxy purine nucleotides or alternately a plurality of purine
nucleotides are 2'-deoxy purine nucleotides).

[0107] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention capable
of mediating RNA interference (RNAi) inside a cell or reconstituted in
vitro system comprising a sense region and an antisense region. In one
embodiment, the sense region comprises one or more 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides),
and one or more 2'-deoxy purine nucleotides (e.g., wherein all purine
nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of
purine nucleotides are 2'-deoxy purine nucleotides). The sense region can
comprise inverted deoxy abasic modifications that are optionally present
at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense
region. The sense region can optionally further comprise a 3'-terminal
overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxyribonucleotides. The antisense region comprises one or more
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately
a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides), and one or more 2'-O-methyl purine nucleotides (e.g.,
wherein all purine nucleotides are 2'-O-methyl purine nucleotides or
alternately a plurality of purine nucleotides are 2'-O-methyl purine
nucleotides). The antisense region can comprise a terminal cap
modification, such as any modification described herein or shown in FIG.
22, that is optionally present at the 3'-end, the 5'-end, or both of the
3' and 5'-ends of the antisense sequence. The antisense region optionally
further comprises a 3'-terminal nucleotide overhang having about 1 to
about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxynucleotides, wherein the
overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or
4) phosphorothioate internucleotide linkages. Non-limiting examples of
these chemically-modified siNAs are shown in FIGS. 18 and 19 and Table IV
herein.

[0108] In another embodiment of the chemically-modified short interfering
nucleic acid comprising a sense region and an antisense region, the sense
region comprises one or more 2'-deoxy-2'-fluoro pyrimidine nucleotides
(e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and one or
more purine ribonucleotides (e.g., wherein all purine nucleotides are
purine ribonucleotides or alternately a plurality of purine nucleotides
are purine ribonucleotides). The sense region can also comprise inverted
deoxy abasic modifications that are optionally present at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the sense region. The sense
region optionally further comprises a 3'-terminal overhang having about 1
to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxyribonucleotides. The
antisense region comprises one or more 2'-deoxy-2'-fluoro pyrimidine
nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides),
and one or more 2'-O-methyl purine nucleotides (e.g., wherein all purine
nucleotides are 2'-O-methyl purine nucleotides or alternately a plurality
of purine nucleotides are 2'-O-methyl purine nucleotides). The antisense
region can also comprise a terminal cap modification, such as any
modification described herein or shown in FIG. 22, that is optionally
present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the
antisense sequence. The antisense region optionally further comprises a
3'-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1,
2, 3, or 4) 2'-deoxynucleotides, wherein the overhang nucleotides can
further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages. Non-limiting examples of these
chemically-modified siNAs are shown in FIGS. 18 and 19 and Table IV
herein.

[0109] In another embodiment of the chemically-modified short interfering
nucleic acid comprising a sense region and an antisense region, the sense
region comprises one or more 2'-deoxy-2'-fluoro pyrimidine nucleotides
(e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and one or
more purine nucleotides selected from the group consisting of 2'-deoxy
nucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl
nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides (e.g.,
wherein all purine nucleotides are selected from the group consisting of
2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,
2'-methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl
nucleotides or alternately a plurality of purine nucleotides are selected
from the group consisting of 2'-deoxy nucleotides, locked nucleic acid
(LNA) nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and
2'-O-methyl nucleotides). The sense region can comprise inverted deoxy
abasic modifications that are optionally present at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the sense region. The sense
region can optionally further comprise a 3'-terminal overhang having
about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxyribonucleotides.
The antisense region comprises one or more 2'-deoxy-2'-fluoro pyrimidine
nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides),
and one or more purine nucleotides selected from the group consisting of
2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,
2'-methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl
nucleotides (e.g., wherein all purine nucleotides are selected from the
group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA)
nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and
2'-O-methyl nucleotides or alternately a plurality of purine nucleotides
are selected from the group consisting of 2'-deoxy nucleotides, locked
nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides,
4'-thionucleotides, and 2'-O-methyl nucleotides). The antisense can also
comprise a terminal cap modification, such as any modification described
herein or shown in FIG. 22, that is optionally present at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the antisense sequence. The
antisense region optionally further comprises a 3'-terminal nucleotide
overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxynucleotides, wherein the overhang nucleotides can further
comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages.

[0110] In another embodiment, any modified nucleotides present in the siNA
molecules of the invention, preferably in the antisense strand of the
siNA molecules of the invention, but also optionally in the sense and/or
both antisense and sense strands, comprise modified nucleotides having
properties or characteristics similar to naturally occurring
ribonucleotides. For example, the invention features siNA molecules
including modified nucleotides having a Northern conformation (e.g.,
Northern pseudorotation cycle, see for example Saenger, Principles of
Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically
modified nucleotides present in the siNA molecules of the invention,
preferably in the antisense strand of the siNA molecules of the
invention, but also optionally in the sense and/or both antisense and
sense strands, are resistant to nuclease degradation while at the same
time maintaining the capacity to mediate RNAi. Non-limiting examples of
nucleotides having a northern configuration include locked nucleic acid
(LNA) nucleotides (e.g., 2'-O,4'-C-methylene-(D-ribofuranosyl)
nucleotides); 2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl,
2'-deoxy-2'-fluoro nucleotides, 2'-deoxy-2'-chloro nucleotides, 2'-azido
nucleotides, and 2'-O-methyl nucleotides.

[0111] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid molecule (siNA) capable of mediating RNA
interference (RNAi) inside a cell or reconstituted in vitro system,
wherein the chemical modification comprises a conjugate attached to the
chemically-modified siNA molecule. The conjugate can be attached to the
chemically-modified siNA molecule via a covalent attachment. In one
embodiment, the conjugate is attached to the chemically-modified siNA
molecule via a biodegradable linker. In one embodiment, the conjugate
molecule is attached at the 3'-end of either the sense strand, the
antisense strand, or both strands of the chemically-modified siNA
molecule. In another embodiment, the conjugate molecule is attached at
the 5'-end of either the sense strand, the antisense strand, or both
strands of the chemically-modified siNA molecule. In yet another
embodiment, the conjugate molecule is attached both the 3'-end and 5'-end
of either the sense strand, the antisense strand, or both strands of the
chemically-modified siNA molecule, or any combination thereof. In one
embodiment, the conjugate molecule of the invention comprises a molecule
that facilitates delivery of a chemically-modified siNA molecule into a
biological system, such as a cell. In another embodiment, the conjugate
molecule attached to the chemically-modified siNA molecule is a poly
ethylene glycol, human serum albumin, or a ligand for a cellular receptor
that can mediate cellular uptake. Examples of specific conjugate
molecules contemplated by the instant invention that can be attached to
chemically-modified siNA molecules are described in Vargeese et al., U.S.
Ser. No. 10/201,394, incorporated by reference herein. The type of
conjugates used and the extent of conjugation of siNA molecules of the
invention can be evaluated for improved pharmacokinetic profiles,
bioavailability, and/or stability of siNA constructs while at the same
time maintaining the ability of the siNA to mediate RNAi activity. As
such, one skilled in the art can screen siNA constructs that are modified
with various conjugates to determine whether the siNA conjugate complex
possesses improved properties while maintaining the ability to mediate
RNAi, for example in animal models as are generally known in the art.

[0112] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention capable
of mediating RNA interference (RNAi) inside a cell or reconstituted in
vitro system, wherein the chemically-modified siNA comprises a sense
region, where one or more pyrimidine nucleotides present in the sense
region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where one or more purine nucleotides present
in the sense region are 2'-deoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-deoxy purine nucleotides or alternately a
plurality of purine nucleotides are 2'-deoxy purine nucleotides), and
inverted deoxy abasic modifications that are optionally present at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense region,
the sense region optionally further comprising a 3'-terminal overhang
having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxyribonucleotides; and wherein the chemically-modified short
interfering nucleic acid molecule comprises an antisense region, where
one or more pyrimidine nucleotides present in the antisense region are
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately
a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides), and wherein one or more purine nucleotides present in the
antisense region are 2'-O-methyl purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl purine nucleotides or alternately a
plurality of purine nucleotides are 2'-O-methyl purine nucleotides), and
a terminal cap modification, such as any modification described herein or
shown in FIG. 22, that is optionally present at the 3'-end, the 5'-end,
or both of the 3' and 5'-ends of the antisense sequence, the antisense
region optionally further comprising a 3'-terminal nucleotide overhang
having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxynucleotides, wherein the overhang nucleotides can further
comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages. Non-limiting examples of these
chemically-modified siNAs are shown in FIGS. 18 and 19 and Table IV
herein.

[0113] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention capable
of mediating RNA interference (RNAi) inside a cell or reconstituted in
vitro system, wherein the chemically-modified siNA comprises a sense
region, where one or more pyrimidine nucleotides present in the sense
region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and where one or more purine nucleotides present
in the sense region are 2'-O-methyl purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl purine nucleotides or alternately a
plurality of purine nucleotides are 2'-O-methyl purine nucleotides), and
inverted deoxy abasic modifications that are optionally present at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense region,
the sense region optionally further comprising a 3'-terminal overhang
having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxyribonucleotides; and wherein the chemically-modified short
interfering nucleic acid molecule comprises an antisense region, where
one or more pyrimidine nucleotides present in the antisense region are
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately
a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides), and wherein one or more purine nucleotides present in the
antisense region are 2'-O-methyl purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl purine nucleotides or alternately a
plurality of purine nucleotides are 2'-O-methyl purine nucleotides), and
a terminal cap modification, such as any modification described herein or
shown in FIG. 22, that is optionally present at the 3'-end, the 5'-end,
or both of the 3' and 5'-ends of the antisense sequence, the antisense
region optionally further comprising a 3'-terminal nucleotide overhang
having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxynucleotides, wherein the overhang nucleotides can further
comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages. Non-limiting examples of these
chemically-modified siNAs are shown in FIGS. 18 and 19 and Table IV
herein.

[0114] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention capable
of mediating RNA interference (RNAi) inside a cell or reconstituted in
vitro system, wherein the siNA comprises a sense region, where one or
more pyrimidine nucleotides present in the sense region are
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately
a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides), and where one or more purine nucleotides present in the
sense region are purine ribonucleotides (e.g., wherein all purine
nucleotides are purine ribonucleotides or alternately a plurality of
purine nucleotides are purine ribonucleotides), and inverted deoxy abasic
modifications that are optionally present at the 3'-end, the 5'-end, or
both of the 3' and 5'-ends of the sense region, the sense region
optionally further comprising a 3'-terminal overhang having about 1 to
about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxyribonucleotides; and wherein
the siNA comprises an antisense region, where one or more pyrimidine
nucleotides present in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides),
and wherein any purine nucleotides present in the antisense region are
2'-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-O-methyl purine nucleotides), and a terminal cap
modification, such as any modification described herein or shown in FIG.
22, that is optionally present at the 3'-end, the 5'-end, or both of the
3' and 5'-ends of the antisense sequence, the antisense region optionally
further comprising a 3'-terminal nucleotide overhang having about 1 to
about 4 (e.g., about 1, 2, 3, or 4) 2'-deoxynucleotides, wherein the
overhang nucleotides can further comprise one or more (e.g., 1, 2, 3, or
4) phosphorothioate internucleotide linkages. Non-limiting examples of
these chemically-modified siNAs are shown in FIGS. 18 and 19 and Table IV
herein.

[0115] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid (siNA) molecule of the invention capable
of mediating RNA interference (RNAi) inside a cell or reconstituted in
vitro system, wherein the chemically-modified siNA comprises a sense
region, where one or more pyrimidine nucleotides present in the sense
region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and for example where one or more purine
nucleotides present in the sense region are selected from the group
consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA)
nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and
2'-O-methyl nucleotides (e.g., wherein all purine nucleotides are
selected from the group consisting of 2'-deoxy nucleotides, locked
nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides,
4'-thionucleotides, and 2'-O-methyl nucleotides or alternately a
plurality of purine nucleotides are selected from the group consisting of
2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,
2'-methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl
nucleotides), and wherein inverted deoxy abasic modifications are
optionally present at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of the sense region, the sense region optionally further
comprising a 3'-terminal overhang having about 1 to about 4 (e.g., about
1, 2, 3, or 4) 2'-deoxyribonucleotides; and wherein the
chemically-modified short interfering nucleic acid molecule comprises an
antisense region, where one or more pyrimidine nucleotides present in the
antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides or alternately a plurality of pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein one or more
purine nucleotides present in the antisense region are selected from the
group consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA)
nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and
2'-O-methyl nucleotides (e.g., wherein all purine nucleotides are
selected from the group consisting of 2'-deoxy nucleotides, locked
nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides,
4'-thionucleotides, and 2'-O-methyl nucleotides or alternately a
plurality of purine nucleotides are selected from the group consisting of
2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,
2'-methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl
nucleotides), and a terminal cap modification, such as any modification
described herein or shown in FIG. 22, that is optionally present at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the antisense
sequence, the antisense region optionally further comprising a
3'-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1,
2, 3, or 4) 2'-deoxynucleotides, wherein the overhang nucleotides can
further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate
internucleotide linkages.

[0116] In one embodiment, the invention features a short interfering
nucleic acid (siNA) molecule of the invention, wherein the siNA further
comprises a nucleotide, non-nucleotide, or mixed
nucleotide/non-nucleotide linker that joins the sense region of the siNA
to the antisense region of the siNA. In one embodiment, a nucleotide
linker of the invention can be a linker of 2 nucleotides in length, for
example 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another
embodiment, the nucleotide linker can be a nucleic acid aptamer. By
"aptamer" or "nucleic acid aptamer" as used herein is meant a nucleic
acid molecule that binds specifically to a target molecule wherein the
nucleic acid molecule has sequence that comprises a sequence recognized
by the target molecule in its natural setting. Alternately, an aptamer
can be a nucleic acid molecule that binds to a target molecule where the
target molecule does not naturally bind to a nucleic acid. The target
molecule can be any molecule of interest. For example, the aptamer can be
used to bind to a ligand-binding domain of a protein, thereby preventing
interaction of the naturally occurring ligand with the protein. This is a
non-limiting example and those in the art will recognize that other
embodiments can be readily generated using techniques generally known in
the art (see, for example, Gold et al., 1995, Annu. Rev. Biochem., 64,
763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin.
Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and
Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry,
45, 1628.)

[0117] In yet another embodiment, a non-nucleotide linker of the invention
comprises abasic nucleotide, polyether, polyamine, polyamide, peptide,
carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.
polyethylene glycols such as those having between 2 and 100 ethylene
glycol units). Specific examples include those described by Seela and
Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,
15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;
Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,
Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand
et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &
Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;
Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International
Publication No. WO 89/02439; Usman et al., International Publication No.
WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and
Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby
incorporated by reference herein. A "non-nucleotide" further means any
group or compound that can be incorporated into a nucleic acid chain in
the place of one or more nucleotide units, including either sugar and/or
phosphate substitutions, and allows the remaining bases to exhibit their
enzymatic activity. The group or compound can be abasic in that it does
not contain a commonly recognized nucleotide base, such as adenosine,
guanine, cytosine, uracil or thymine, for example at the Cl position of
the sugar.

[0118] In one embodiment, the invention features a chemically-modified
short interfering nucleic acid molecule (siNA) capable of mediating RNA
interference (RNAi) against a target gene inside a cell or reconstituted
in vitro system, wherein the chemical modification comprises a conjugate
covalently attached to the chemically-modified siNA molecule.
Non-limiting examples of conjugates contemplated by the invention include
conjugates and ligands described in Vargeese et al., U.S. Ser. No.
10/427,160, filed Apr. 30, 2003, incorporated by reference herein in its
entirety, including the drawings. In another embodiment, the conjugate is
covalently attached to the chemically-modified siNA molecule via a
biodegradable linker. In one embodiment, the conjugate molecule is
attached at the 3'-end of either the sense strand, the antisense strand,
or both strands of the chemically-modified siNA molecule. In another
embodiment, the conjugate molecule is attached at the 5'-end of either
the sense strand, the antisense strand, or both strands of the
chemically-modified siNA molecule. In yet another embodiment, the
conjugate molecule is attached both the 3'-end and 5'-end of either the
sense strand, the antisense strand, or both strands of the
chemically-modified siNA molecule, or any combination thereof. In one
embodiment, a conjugate molecule of the invention comprises a molecule
that facilitates delivery of a chemically-modified siNA molecule into a
biological system, such as a cell. In another embodiment, the conjugate
molecule attached to the chemically-modified siNA molecule is a
polyethylene glycol, human serum albumin, or a ligand for a cellular
receptor that can mediate cellular uptake. Examples of specific conjugate
molecules contemplated by the instant invention that can be attached to
chemically-modified siNA molecules are described in Vargeese et al., U.S.
Ser. No. 10/201,394, incorporated by reference herein. The type of
conjugates used and the extent of conjugation of siNA molecules of the
invention can be evaluated for improved pharmacokinetic profiles,
bioavailability, and/or stability of siNA constructs while at the same
time maintaining the ability of the siNA to mediate RNAi activity. As
such, one skilled in the art can screen siNA constructs that are modified
with various conjugates to determine whether the siNA conjugate complex
possesses improved properties while maintaining the ability to mediate
RNAi, for example in animal models as are generally known in the art.

[0119] In one embodiment, the invention features a short interfering
nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi)
inside a cell or reconstituted in vitro system, wherein one or both
strands of the siNA molecule that are assembled from two separate
oligonucleotides do not comprise any ribonucleotides. For example, a siNA
molecule can be assembled from a single oligonucleotide where the sense
and antisense regions of the siNA comprise separate oligonucleotides that
do not have any ribonucleotides (e.g., nucleotides having a 2'-OH group)
present in the oligonucleotides. In another example, a siNA molecule can
be assembled from a single oligonucleotide where the sense and antisense
regions of the siNA are linked or circularized by a nucleotide or
non-nucleotide linker as described herein, wherein the oligonucleotide
does not have any ribonucleotides (e.g., nucleotides having a 2'-OH
group) present in the oligonucleotide. Applicant has surprisingly found
that the presence of ribonucleotides (e.g., nucleotides having a
2'-hydroxyl group) within the siNA molecule is not required or essential
to support RNAi activity. As such, in one embodiment, all positions
within the siNA can include chemically modified nucleotides and/or
non-nucleotides such as nucleotides and or non-nucleotides having Formula
I, II, III, IV, V, VI, or VII or any combination thereof to the extent
that the ability of the siNA molecule to support RNAi activity in a cell
is maintained.

[0120] In one embodiment, the invention features a siNA molecule that does
not require the presence of a 2'-OH group (ribonucleotide) to be present
withing the siNA molecule to support RNA interference.

[0121] In one embodiment, a siNA molecule of the invention is a single
stranded siNA molecule that mediates RNAi activity in a cell or
reconstituted in vitro system, wherein the siNA molecule comprises a
single stranded polynucleotide having complementarity to a target nucleic
acid sequence. In another embodiment, the single stranded siNA molecule
of the invention comprises a 5'-terminal phosphate group. In another
embodiment, the single stranded siNA molecule of the invention comprises
a 5'-terminal phosphate group and a 3'-terminal phosphate group (e.g., a
2',3'-cyclic phosphate). In another embodiment, the single stranded siNA
molecule of the invention comprises about 19 to about 29 (e.g., about 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In yet another
embodiment, the single stranded siNA molecule of the invention comprises
one or more chemically modified nucleotides or non-nucleotides described
herein. For example, all the positions within the siNA molecule can
include chemically-modified nucleotides such as nucleotides having any of
Formulae I-VII, or any combination thereof to the extent that the ability
of the siNA molecule to support RNAi activity in a cell is maintained.

[0122] In one embodiment, the single stranded siNA molecule having
complementarity to a target nucleic acid sequence comprises one or more
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately
a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides), and one or more 2'-O-methyl purine nucleotides (e.g.,
wherein all purine nucleotides are 2'-O-methyl purine nucleotides or
alternately a plurality of purine nucleotides are 2'-O-methyl purine
nucleotides). In another embodiment, the single stranded siNA molecule
comprises one or more 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides or alternately a plurality of pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and one or more 2'-deoxy
purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy
purine nucleotides or alternately a plurality of purine nucleotides are
2'-deoxy purine nucleotides). In another embodiment, the single stranded
siNA molecule comprises one or more 2'-deoxy-2'-fluoro pyrimidine
nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides),
wherein any purine nucleotides present in the antisense region are locked
nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are
LNA nucleotides or alternately a plurality of purine nucleotides are LNA
nucleotides). In another embodiment, the single stranded siNA molecule
comprises one or more 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g.,
wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides or alternately a plurality of pyrimidine nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and one or more
2'-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides
are 2'-methoxyethyl purine nucleotides or alternately a plurality of
purine nucleotides are 2'-methoxyethyl purine nucleotides), the single
stranded siNA can comprise a terminal cap modification, such as any
modification described herein or shown in FIG. 22, that is optionally
present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the
antisense sequence. The single stranded siNA optionally further comprises
about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal
2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the
terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or
4) phosphorothioate internucleotide linkages. The single stranded siNA
optionally further comprises a terminal phosphate group, such as a
5'-terminal phosphate group.

[0123] In one embodiment, a siNA molecule of the invention is a single
stranded siNA molecule that mediates RNAi activity in a cell or
reconstituted in vitro system, wherein the siNA molecule comprises a
single stranded polynucleotide having complementarity to a target nucleic
acid sequence, and wherein one or more pyrimidine nucleotides present in
the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present in
the antisense region are 2'-O-methyl purine nucleotides (e.g., wherein
all purine nucleotides are 2'-O-methyl purine nucleotides or alternately
a plurality of purine nucleotides are 2'-O-methyl purine nucleotides),
and a terminal cap modification, such as any modification described
herein or shown in FIG. 22, that is optionally present at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the antisense sequence. The siNA
optionally further comprises about 1 to about 4 or more (e.g., about 1,
2, 3, 4 or more) terminal 2'-deoxynucleotides at the 3'-end of the siNA
molecule, wherein the terminal nucleotides can further comprise one or
more (e.g., 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate,
and/or thiophosphonoacetate internucleotide linkages, and wherein the
siNA optionally further comprises a terminal phosphate group, such as a
5'-terminal phosphate group. In any of these embodiments, any purine
nucleotides present in the antisense region are alternatively 2'-deoxy
purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy
purine nucleotides or alternately a plurality of purine nucleotides are
2'-deoxy purine nucleotides). Also, in any of these embodiments, any
purine nucleotides present in the siNA (i.e., purine nucleotides present
in the sense and/or antisense region) can alternatively be locked nucleic
acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA
nucleotides or alternately a plurality of purine nucleotides are LNA
nucleotides). Also, in any of these embodiments, any purine nucleotides
present in the siNA are alternatively 2'-methoxyethyl purine nucleotides
(e.g., wherein all purine nucleotides are 2'-methoxyethyl purine
nucleotides or alternately a plurality of purine nucleotides are
2'-methoxyethyl purine nucleotides). In another embodiment, any modified
nucleotides present in the single stranded siNA molecules of the
invention comprise modified nucleotides having properties or
characteristics similar to naturally occurring ribonucleotides. For
example, the invention features siNA molecules including modified
nucleotides having a Northern conformation (e.g., Northern pseudorotation
cycle, see for example Saenger, Principles of Nucleic Acid Structure,
Springer-Verlag ed., 1984). As such, chemically modified nucleotides
present in the single stranded siNA molecules of the invention are
preferably resistant to nuclease degradation while at the same time
maintaining the capacity to mediate RNAi

[0124] In one embodiment, a siNA molecule of the invention is a single
stranded siNA molecule that mediates RNAi activity in a cell or
reconstituted in vitro system, wherein the siNA molecule comprises a
single stranded polynucleotide having complementarity to a target nucleic
acid sequence, and wherein one or more pyrimidine nucleotides present in
the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present in
the siNA are 2'-O-methyl purine nucleotides (e.g., wherein all purine
nucleotides are 2'-O-methyl purine nucleotides or alternately a plurality
of purine nucleotides are 2'-O-methyl purine nucleotides), and a terminal
cap modification, such as any modification described herein or shown in
FIG. 22, that is optionally present at the 3'-end, the 5'-end, or both of
the 3' and 5'-ends of the antisense sequence, the siNA optionally further
comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal
2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the
terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or
4) phosphorothioate internucleotide linkages, and wherein the siNA
optionally further comprises a terminal phosphate group, such as a
5'-terminal phosphate group.

[0125] In one embodiment, a siNA molecule of the invention is a single
stranded siNA molecule that mediates RNAi activity in a cell or
reconstituted in vitro system, wherein the siNA molecule comprises a
single stranded polynucleotide having complementarity to a target nucleic
acid sequence, and wherein one or more pyrimidine nucleotides present in
the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present in
the siNA are 2'-deoxy purine nucleotides (e.g., wherein all purine
nucleotides are 2'-deoxy purine nucleotides or alternately a plurality of
purine nucleotides are 2'-deoxy purine nucleotides), and a terminal cap
modification, such as any modification described herein or shown in FIG.
22, that is optionally present at the 3'-end, the 5'-end, or both of the
3' and 5'-ends of the antisense sequence, the siNA optionally further
comprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal
2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the
terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or
4) phosphorothioate internucleotide linkages, and wherein the siNA
optionally further comprises a terminal phosphate group, such as a
5'-terminal phosphate group.

[0126] In one embodiment, a siNA molecule of the invention is a single
stranded siNA molecule that mediates RNAi activity in a cell or
reconstituted in vitro system, wherein the siNA molecule comprises a
single stranded polynucleotide having complementarity to a target nucleic
acid sequence, and wherein one or more pyrimidine nucleotides present in
the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present in
the siNA are locked nucleic acid (LNA) nucleotides (e.g., wherein all
purine nucleotides are LNA nucleotides or alternately a plurality of
purine nucleotides are LNA nucleotides), and a terminal cap modification,
such as any modification described herein or shown in FIG. 22, that is
optionally present at the 3'-end, the 5'-end, or both of the 3' and
5'-ends of the antisense sequence, the siNA optionally further comprising
about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal
2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the
terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or
4) phosphorothioate internucleotide linkages, and wherein the siNA
optionally further comprises a terminal phosphate group, such as a
5'-terminal phosphate group.

[0127] In one embodiment, a siNA molecule of the invention is a single
stranded siNA molecule that mediates RNAi activity in a cell or
reconstituted in vitro system, wherein the siNA molecule comprises a
single stranded polynucleotide having complementarity to a target nucleic
acid sequence, and wherein one or more pyrimidine nucleotides present in
the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or
alternately a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro
pyrimidine nucleotides), and wherein any purine nucleotides present in
the siNA are 2'-methoxyethyl purine nucleotides (e.g., wherein all purine
nucleotides are 2'-methoxyethyl purine nucleotides or alternately a
plurality of purine nucleotides are 2'-methoxyethyl purine nucleotides),
and a terminal cap modification, such as any modification described
herein or shown in FIG. 22, that is optionally present at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the antisense sequence, the siNA
optionally further comprising about 1 to about 4 (e.g., about 1, 2, 3, or
4) terminal 2'-deoxynucleotides at the 3'-end of the siNA molecule,
wherein the terminal nucleotides can further comprise one or more (e.g.,
1, 2, 3, or 4) phosphorothioate internucleotide linkages, and wherein the
siNA optionally further comprises a terminal phosphate group, such as a
5'-terminal phosphate group.

[0128] In another embodiment, any modified nucleotides present in the
single stranded siNA molecules of the invention comprise modified
nucleotides having properties or characteristics similar to naturally
occurring ribonucleotides. For example, the invention features siNA
molecules including modified nucleotides having a Northern conformation
(e.g., Northern pseudorotation cycle, see for example Saenger, Principles
of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such,
chemically modified nucleotides present in the single stranded siNA
molecules of the invention are preferably resistant to nuclease
degradation while at the same time maintaining the capacity to mediate
RNAi.

[0129] In one embodiment, the invention features a method for modulating
the expression of a gene within a cell comprising: (a) synthesizing a
siNA molecule of the invention, which can be chemically-modified, wherein
one of the siNA strands comprises a sequence complementary to RNA of the
gene; and (b) introducing the siNA molecule into a cell under conditions
suitable to modulate the expression of the gene in the cell.

[0130] In one embodiment, the invention features a method for modulating
the expression of a gene within a cell comprising: (a) synthesizing a
siNA molecule of the invention, which can be chemically-modified, wherein
one of the siNA strands comprises a sequence complementary to RNA of the
gene and wherein the sense strand sequence of the siNA comprises a
sequence substantially similar to the sequence of the target RNA; and (b)
introducing the siNA molecule into a cell under conditions suitable to
modulate the expression of the gene in the cell.

[0131] In another embodiment, the invention features a method for
modulating the expression of more than one gene within a cell comprising:
(a) synthesizing siNA molecules of the invention, which can be
chemically-modified, wherein one of the siNA strands comprises a sequence
complementary to RNA of the genes; and (b) introducing the siNA molecules
into a cell under conditions suitable to modulate the expression of the
genes in the cell.

[0132] In another embodiment, the invention features a method for
modulating the expression of more than one gene within a cell comprising:
(a) synthesizing a siNA molecule of the invention, which can be
chemically-modified, wherein one of the siNA strands comprises a sequence
complementary to RNA of the gene and wherein the sense strand sequence of
the siNA comprises a sequence substantially similar to the sequence of
the target RNA; and (b) introducing the siNA molecules into a cell under
conditions suitable to modulate the expression of the genes in the cell.

[0133] In one embodiment, siNA molecules of the invention are used as
reagents in ex vivo applications. For example, siNA reagents are
introduced into tissue or cells that are transplanted into a subject for
therapeutic effect. The cells and/or tissue can be derived from an
organism or subject that later receives the explant, or can be derived
from another organism or subject prior to transplantation. The siNA
molecules can be used to modulate the expression of one or more genes in
the cells or tissue, such that the cells or tissue obtain a desired
phenotype or are able to perform a function when transplanted in vivo. In
one embodiment, certain target cells from a patient are extracted. These
extracted cells are contacted with siNAs targeting a specific nucleotide
sequence within the cells under conditions suitable for uptake of the
siNAs by these cells (e.g. using delivery reagents such as cationic
lipids, liposomes and the like or using techniques such as
electroporation to facilitate the delivery of siNAs into cells). The
cells are then reintroduced back into the same patient or other patients.
Non-limiting examples of ex vivo applications include use in organ/tissue
transplant, tissue grafting, or treatment of pulmonary disease (e.g.,
restenosis) or prevent neointimal hyperplasia and atherosclerosis in vein
grafts. Such ex vivo applications may also used to treat conditions
associated with coronary and peripheral bypass graft failure, for
example, such methods can be used in conjunction with peripheral vascular
bypass graft surgery and coronary artery bypass graft surgery. Additional
applications include transplants to treat CNS lesions or injury,
including use in treatment of neurodegenerative conditions such as
Alzheimer's disease, Parkinson's Disease, Epilepsy, Dementia,
Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0134] In one embodiment, the invention features a method of modulating
the expression of a gene in a tissue explant comprising: (a) synthesizing
a siNA molecule of the invention, which can be chemically-modified,
wherein one of the siNA strands comprises a sequence complementary to RNA
of the gene; and (b) introducing the siNA molecule into a cell of the
tissue explant derived from a particular organism under conditions
suitable to modulate the expression of the gene in the tissue explant. In
another embodiment, the method further comprises introducing the tissue
explant back into the organism the tissue was derived from or into
another organism under conditions suitable to modulate the expression of
the gene in that organism.

[0135] In one embodiment, the invention features a method of modulating
the expression of a gene in a tissue explant comprising: (a) synthesizing
a siNA molecule of the invention, which can be chemically-modified,
wherein one of the siNA strands comprises a sequence complementary to RNA
of the gene and wherein the sense strand sequence of the siNA comprises a
sequence substantially similar to the sequence of the target RNA; and (b)
introducing the siNA molecule into a cell of the tissue explant derived
from a particular organism under conditions suitable to modulate the
expression of the gene in the tissue explant. In another embodiment, the
method further comprises introducing the tissue explant back into the
organism the tissue was derived from or into another organism under
conditions suitable to modulate the expression of the gene in that
organism.

[0136] In another embodiment, the invention features a method of
modulating the expression of more than one gene in a tissue explant
comprising: (a) synthesizing siNA molecules of the invention, which can
be chemically-modified, wherein one of the siNA strands comprises a
sequence complementary to RNA of the genes; and (b) introducing the siNA
molecules into a cell of the tissue explant derived from a particular
organism under conditions suitable to modulate the expression of the
genes in the tissue explant. In another embodiment, the method further
comprises introducing the tissue explant back into the organism the
tissue was derived from or into another organism under conditions
suitable to modulate the expression of the genes in that organism.

[0137] In one embodiment, the invention features a method of modulating
the expression of a gene in an organism comprising: (a) synthesizing a
siNA molecule of the invention, which can be chemically-modified, wherein
one of the siNA strands comprises a sequence complementary to RNA of the
gene; and (b) introducing the siNA molecule into the organism under
conditions suitable to modulate the expression of the gene in the
organism.

[0138] In another embodiment, the invention features a method of
modulating the expression of more than one gene in an organism
comprising: (a) synthesizing siNA molecules of the invention, which can
be chemically-modified, wherein one of the siNA strands comprises a
sequence complementary to RNA of the genes; and (b) introducing the siNA
molecules into the organism under conditions suitable to modulate the
expression of the genes in the organism.

[0139] In one embodiment, the invention features a method for modulating
the expression of a gene within a cell comprising: (a) synthesizing a
siNA molecule of the invention, which can be chemically-modified, wherein
the siNA comprises a single stranded sequence having complementarity to
RNA of the gene; and (b) introducing the siNA molecule into a cell under
conditions suitable to modulate the expression of the gene in the cell.

[0140] In one embodiment, the invention features a method of modulating
the expression of a target gene in an tissue or organ comprising: (a)
synthesizing a siNA molecule of the invention, which can be
chemically-modified, wherein the siNA comprises a single stranded
sequence having complementarity to RNA of the target gene; and (b)
introducing the siNA molecule into the tissue or organ under conditions
suitable to modulate the expression of the target gene in the organism.
In another embodiment, the tissue is ocular tissue and the organ is the
eye. In another embodiment, the tissue comprises hepatocytes and/or
hepatic tissue and the organ is the liver.

[0141] In another embodiment, the invention features a method for
modulating the expression of more than one gene within a cell comprising:
(a) synthesizing siNA molecules of the invention, which can be
chemically-modified, wherein the siNA comprises a single stranded
sequence having complementarity to RNA of the gene; and (b) contacting
the siNA molecule with a cell in vitro or in vivo under conditions
suitable to modulate the expression of the genes in the cell.

[0142] In one embodiment, the invention features a method of modulating
the expression of a gene in a tissue explant comprising: (a) synthesizing
a siNA molecule of the invention, which can be chemically-modified,
wherein the siNA comprises a single stranded sequence having
complementarity to RNA of the gene; and (b) contacting the siNA molecule
with a cell of the tissue explant derived from a particular organism
under conditions suitable to modulate the expression of the gene in the
tissue explant. In another embodiment, the method further comprises
introducing the tissue explant back into the organism the tissue was
derived from or into another organism under conditions suitable to
modulate the expression of the gene in that organism.

[0143] In another embodiment, the invention features a method of
modulating the expression of more than one gene in a tissue explant
comprising: (a) synthesizing siNA molecules of the invention, which can
be chemically-modified, wherein the siNA comprises a single stranded
sequence having complementarity to RNA of the gene; and (b) introducing
the siNA molecules into a cell of the tissue explant derived from a
particular organism under conditions suitable to modulate the expression
of the genes in the tissue explant. In another embodiment, the method
further comprises introducing the tissue explant back into the organism
the tissue was derived from or into another organism under conditions
suitable to modulate the expression of the genes in that organism.

[0144] In one embodiment, the invention features a method of modulating
the expression of a gene in an organism comprising: (a) synthesizing a
siNA molecule of the invention, which can be chemically-modified, wherein
the siNA comprises a single stranded sequence having complementarity to
RNA of the gene; and (b) introducing the siNA molecule into the organism
under conditions suitable to modulate the expression of the gene in the
organism.

[0145] In another embodiment, the invention features a method of
modulating the expression of more than one gene in an organism
comprising: (a) synthesizing siNA molecules of the invention, which can
be chemically-modified, wherein the siNA comprises a single stranded
sequence having complementarity to RNA of the gene; and (b) introducing
the siNA molecules into the organism under conditions suitable to
modulate the expression of the genes in the organism.

[0146] In one embodiment, the invention features a method of modulating
the expression of a gene in an organism comprising contacting the
organism with a siNA molecule of the invention under conditions suitable
to modulate the expression of the gene in the organism.

[0147] In another embodiment, the invention features a method of
modulating the expression of more than one gene in an organism comprising
contacting the organism with one or more siNA molecules of the invention
under conditions suitable to modulate the expression of the genes in the
organism.

[0148] The siNA molecules of the invention can be designed to down
regulate or inhibit target gene expression through RNAi targeting of a
variety of RNA molecules. In one embodiment, the siNA molecules of the
invention are used to target various RNAs corresponding to a target gene.
Non-limiting examples of such RNAs include messenger RNA (mRNA),
alternate RNA splice variants of target gene(s), post-transcriptionally
modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA
templates. If alternate splicing produces a family of transcripts that
are distinguished by usage of appropriate exons, the instant invention
can be used to inhibit gene expression through the appropriate exons to
specifically inhibit or to distinguish among the functions of gene family
members. For example, a protein that contains an alternatively spliced
transmembrane domain can be expressed in both membrane bound and secreted
forms. Use of the invention to target the exon containing the
transmembrane domain can be used to determine the functional consequences
of pharmaceutical targeting of membrane bound as opposed to the secreted
form of the protein. Non-limiting examples of applications of the
invention relating to targeting these RNA molecules include therapeutic
pharmaceutical applications, pharmaceutical discovery applications,
molecular diagnostic and gene function applications, and gene mapping,
for example using single nucleotide polymorphism mapping with siNA
molecules of the invention. Such applications can be implemented using
known gene sequences or from partial sequences available from an
expressed sequence tag (EST).

[0149] In another embodiment, the siNA molecules of the invention are used
to target conserved sequences corresponding to a gene family or gene
families. As such, siNA molecules targeting multiple gene targets can
provide increased therapeutic effect. In addition, siNA can be used to
characterize pathways of gene function in a variety of applications. For
example, the present invention can be used to inhibit the activity of
target gene(s) in a pathway to determine the function of uncharacterized
gene(s) in gene function analysis, mRNA function analysis, or
translational analysis. The invention can be used to determine potential
target gene pathways involved in various diseases and conditions toward
pharmaceutical development. The invention can be used to understand
pathways of gene expression involved in, for example, in development,
such as prenatal development and postnatal development, and/or the
progression and/or maintenance of cancer, infectious disease,
autoimmunity, inflammation, endocrine disorders, renal disease, pulmonary
disease, cardiovascular disease, birth defects, ageing, any other disease
or condition related to gene expression.

[0150] In one embodiment, siNA molecule(s) and/or methods of the invention
are used to down-regulate or inhibit the expression of gene(s) that
encode RNA referred to by Genbank Accession, for example genes encoding
RNA sequence(s) referred to herein by Genbank Accession number.

[0151] In one embodiment, the invention features a method comprising: (a)
generating a library of siNA constructs having a predetermined
complexity; and (b) assaying the siNA constructs of (a) above, under
conditions suitable to determine RNAi target sites within the target RNA
sequence. In one embodiment, the siNA molecules of (a) have strands of a
fixed length, for example, about 23 nucleotides in length. In another
embodiment, the siNA molecules of (a) are of differing length, for
example having strands of about 19 to about 25 (e.g., about 19, 20, 21,
22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay
can comprise a reconstituted in vitro siNA assay as described herein. In
another embodiment, the assay can comprise a cell culture system in which
target RNA is expressed. In another embodiment, fragments of target RNA
are analyzed for detectable levels of cleavage, for example by gel
electrophoresis, northern blot analysis, or RNAse protection assays, to
determine the most suitable target site(s) within the target RNA
sequence. The target RNA sequence can be obtained as is known in the art,
for example, by cloning and/or transcription for in vitro systems, and by
cellular expression in in vivo systems.

[0152] In one embodiment, the invention features a method comprising: (a)
generating a randomized library of siNA constructs having a predetermined
complexity, such as of 4N, where N represents the number of base paired
nucleotides in each of the siNA construct strands (eg. for a siNA
construct having 21 nucleotide sense and antisense strands with 19 base
pairs, the complexity would be 419); and (b) assaying the siNA
constructs of (a) above, under conditions suitable to determine RNAi
target sites within the target RNA sequence. In another embodiment, the
siNA molecules of (a) have strands of a fixed length, for example about
23 nucleotides in length. In yet another embodiment, the siNA molecules
of (a) are of differing length, for example having strands of about 19 to
about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in
length. In one embodiment, the assay can comprise a reconstituted in
vitro siNA assay as described in Example 7 herein. In another embodiment,
the assay can comprise a cell culture system in which target RNA is
expressed. In another embodiment, fragments of target RNA are analyzed
for detectable levels of cleavage, for example by gel electrophoresis,
northern blot analysis, or RNAse protection assays, to determine the most
suitable target site(s) within the target RNA sequence. In another
embodiment, the target RNA sequence can be obtained as is known in the
art, for example, by cloning and/or transcription for in vitro systems,
and by cellular expression in in vivo systems.

[0153] In another embodiment, the invention features a method comprising:
(a) analyzing the sequence of a RNA target encoded by a target gene; (b)
synthesizing one or more sets of siNA molecules having sequence
complementary to one or more regions of the RNA of (a); and (c) assaying
the siNA molecules of (b) under conditions suitable to determine RNAi
targets within the target RNA sequence. In one embodiment, the siNA
molecules of (b) have strands of a fixed length, for example about 23
nucleotides in length. In another embodiment, the siNA molecules of (b)
are of differing length, for example having strands of about 19 to about
25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In
one embodiment, the assay can comprise a reconstituted in vitro siNA
assay as described herein. In another embodiment, the assay can comprise
a cell culture system in which target RNA is expressed. Fragments of
target RNA are analyzed for detectable levels of cleavage, for example by
gel electrophoresis, northern blot analysis, or RNAse protection assays,
to determine the most suitable target site(s) within the target RNA
sequence. The target RNA sequence can be obtained as is known in the art,
for example, by cloning and/or transcription for in vitro systems, and by
expression in in vivo systems.

[0154] By "target site" is meant a sequence within a target RNA that is
"targeted" for cleavage mediated by a siNA construct which contains
sequences within its antisense region that are complementary to the
target sequence.

[0155] By "detectable level of cleavage" is meant cleavage of target RNA
(and formation of cleaved product RNAs) to an extent sufficient to
discern cleavage products above the background of RNAs produced by random
degradation of the target RNA. Production of cleavage products from 1-5%
of the target RNA is sufficient to detect above the background for most
methods of detection.

[0156] In one embodiment, the invention features a composition comprising
a siNA molecule of the invention, which can be chemically-modified, in a
pharmaceutically acceptable carrier or diluent. In another embodiment,
the invention features a pharmaceutical composition comprising siNA
molecules of the invention, which can be chemically-modified, targeting
one or more genes in a pharmaceutically acceptable carrier or diluent. In
another embodiment, the invention features a method for diagnosing a
disease or condition in a subject comprising administering to the subject
a composition of the invention under conditions suitable for the
diagnosis of the disease or condition in the subject. In another
embodiment, the invention features a method for treating or preventing a
disease or condition in a subject, comprising administering to the
subject a composition of the invention under conditions suitable for the
treatment or prevention of the disease or condition in the subject, alone
or in conjunction with one or more other therapeutic compounds. In yet
another embodiment, the invention features a method for reducing or
preventing tissue rejection in a subject comprising administering to the
subject a composition of the invention under conditions suitable for the
reduction or prevention of tissue rejection in the subject.

[0157] In another embodiment, the invention features a method for
validating a gene target, comprising: (a) synthesizing a siNA molecule of
the invention, which can be chemically-modified, wherein one of the siNA
strands includes a sequence complementary to RNA of a target gene; (b)
introducing the siNA molecule into a cell, tissue, or organism under
conditions suitable for modulating expression of the target gene in the
cell, tissue, or organism; and (c) determining the function of the gene
by assaying for any phenotypic change in the cell, tissue, or organism.

[0158] In another embodiment, the invention features a method for
validating a target gene comprising: (a) synthesizing a siNA molecule of
the invention, which can be chemically-modified, wherein one of the siNA
strands includes a sequence complementary to RNA of a target gene; (b)
introducing the siNA molecule into a biological system under conditions
suitable for modulating expression of the target gene in the biological
system; and (c) determining the function of the gene by assaying for any
phenotypic change in the biological system.

[0159] By "biological system" is meant, material, in a purified or
unpurified form, from biological sources, including but not limited to
human, animal, plant, insect, bacterial, viral or other sources, wherein
the system comprises the components required for RNAi activity. The term
"biological system" includes, for example, a cell, tissue, or organism,
or extract thereof. The term biological system also includes
reconstituted RNAi systems that can be used in an in vitro setting.

[0160] By "phenotypic change" is meant any detectable change to a cell
that occurs in response to contact or treatment with a nucleic acid
molecule of the invention (e.g., siNA). Such detectable changes include,
but are not limited to, changes in shape, size, proliferation, motility,
protein expression or RNA expression or other physical or chemical
changes as can be assayed by methods known in the art. The detectable
change can also include expression of reporter genes/molecules such as
Green Florescent Protein (GFP) or various tags that are used to identify
an expressed protein or any other cellular component that can be assayed.

[0161] In one embodiment, the invention features a kit containing a siNA
molecule of the invention, which can be chemically-modified, that can be
used to modulate the expression of a target gene in biological system,
including, for example, in a cell, tissue, or organism. In another
embodiment, the invention features a kit containing more than one siNA
molecule of the invention, which can be chemically-modified, that can be
used to modulate the expression of more than one target gene in a
biological system, including, for example, in a cell, tissue, or
organism.

[0162] In one embodiment, the invention features a kit containing a siNA
molecule of the invention, which can be chemically-modified, that can be
used to modulate the expression of a target gene in a biological system.
In another embodiment, the invention features a kit containing more than
one siNA molecule of the invention, which can be chemically-modified,
that can be used to modulate the expression of more than one target gene
in a biological system.

[0163] In one embodiment, the invention features a cell containing one or
more siNA molecules of the invention, which can be chemically-modified.
In another embodiment, the cell containing a siNA molecule of the
invention is a mammalian cell. In yet another embodiment, the cell
containing a siNA molecule of the invention is a human cell.

[0164] In one embodiment, the synthesis of a siNA molecule of the
invention, which can be chemically-modified, comprises: (a) synthesis of
two complementary strands of the siNA molecule; (b) annealing the two
complementary strands together under conditions suitable to obtain a
double-stranded siNA molecule. In another embodiment, synthesis of the
two complementary strands of the siNA molecule is by solid phase
oligonucleotide synthesis. In yet another embodiment, synthesis of the
two complementary strands of the siNA molecule is by solid phase tandem
oligonucleotide synthesis.

[0165] In one embodiment, the invention features a method for synthesizing
a siNA duplex molecule comprising: (a) synthesizing a first
oligonucleotide sequence strand of the siNA molecule, wherein the first
oligonucleotide sequence strand comprises a cleavable linker molecule
that can be used as a scaffold for the synthesis of the second
oligonucleotide sequence strand of the siNA; (b) synthesizing the second
oligonucleotide sequence strand of siNA on the scaffold of the first
oligonucleotide sequence strand, wherein the second oligonucleotide
sequence strand further comprises a chemical moiety than can be used to
purify the siNA duplex; (c) cleaving the linker molecule of (a) under
conditions suitable for the two siNA oligonucleotide strands to hybridize
and form a stable duplex; and (d) purifying the siNA duplex utilizing the
chemical moiety of the second oligonucleotide sequence strand. In one
embodiment, cleavage of the linker molecule in (c) above takes place
during deprotection of the oligonucleotide, for example, under hydrolysis
conditions using an alkylamine base such as methylamine. In one
embodiment, the method of synthesis comprises solid phase synthesis on a
solid support such as controlled pore glass (CPG) or polystyrene, wherein
the first sequence of (a) is synthesized on a cleavable linker, such as a
succinyl linker, using the solid support as a scaffold. The cleavable
linker in (a) used as a scaffold for synthesizing the second strand can
comprise similar reactivity as the solid support derivatized linker, such
that cleavage of the solid support derivatized linker and the cleavable
linker of (a) takes place concomitantly. In another embodiment, the
chemical moiety of (b) that can be used to isolate the attached
oligonucleotide sequence comprises a trityl group, for example a
dimethoxytrityl group, which can be employed in a trityl-on synthesis
strategy as described herein. In yet another embodiment, the chemical
moiety, such as a dimethoxytrityl group, is removed during purification,
for example, using acidic conditions.

[0166] In a further embodiment, the method for siNA synthesis is a
solution phase synthesis or hybrid phase synthesis wherein both strands
of the siNA duplex are synthesized in tandem using a cleavable linker
attached to the first sequence which acts a scaffold for synthesis of the
second sequence. Cleavage of the linker under conditions suitable for
hybridization of the separate siNA sequence strands results in formation
of the double-stranded siNA molecule.

[0167] In another embodiment, the invention features a method for
synthesizing a siNA duplex molecule comprising: (a) synthesizing one
oligonucleotide sequence strand of the siNA molecule, wherein the
sequence comprises a cleavable linker molecule that can be used as a
scaffold for the synthesis of another oligonucleotide sequence; (b)
synthesizing a second oligonucleotide sequence having complementarity to
the first sequence strand on the scaffold of (a), wherein the second
sequence comprises the other strand of the double-stranded siNA molecule
and wherein the second sequence further comprises a chemical moiety than
can be used to isolate the attached oligonucleotide sequence; (c)
purifying the product of (b) utilizing the chemical moiety of the second
oligonucleotide sequence strand under conditions suitable for isolating
the full-length sequence comprising both siNA oligonucleotide strands
connected by the cleavable linker and under conditions suitable for the
two siNA oligonucleotide strands to hybridize and form a stable duplex.
In one embodiment, cleavage of the linker molecule in (c) above takes
place during deprotection of the oligonucleotide, for example under
hydrolysis conditions. In another embodiment, cleavage of the linker
molecule in (c) above takes place after deprotection of the
oligonucleotide. In another embodiment, the method of synthesis comprises
solid phase synthesis on a solid support such as controlled pore glass
(CPG) or polystyrene, wherein the first sequence of (a) is synthesized on
a cleavable linker, such as a succinyl linker, using the solid support as
a scaffold. The cleavable linker in (a) used as a scaffold for
synthesizing the second strand can comprise similar reactivity or
differing reactivity as the solid support derivatized linker, such that
cleavage of the solid support derivatized linker and the cleavable linker
of (a) takes place either concomitantly or sequentially. In one
embodiment, the chemical moiety of (b) that can be used to isolate the
attached oligonucleotide sequence comprises a trityl group, for example a
dimethoxytrityl group.

[0168] In another embodiment, the invention features a method for making a
double-stranded siNA molecule in a single synthetic process comprising:
(a) synthesizing an oligonucleotide having a first and a second sequence,
wherein the first sequence is complementary to the second sequence, and
the first oligonucleotide sequence is linked to the second sequence via a
cleavable linker, and wherein a terminal 5'-protecting group, for
example, a 5'-O-dimethoxytrityl group (5'-O-DMT) remains on the
oligonucleotide having the second sequence; (b) deprotecting the
oligonucleotide whereby the deprotection results in the cleavage of the
linker joining the two oligonucleotide sequences; and (c) purifying the
product of (b) under conditions suitable for isolating the
double-stranded siNA molecule, for example using a trityl-on synthesis
strategy as described herein.

[0169] In another embodiment, the method of synthesis of siNA molecules of
the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos.
5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in
their entirety.

[0170] In one embodiment, the invention features siNA constructs that
mediate RNAi in a cell or reconstituted system, wherein the siNA
construct comprises one or more chemical modifications, for example, one
or more chemical modifications having any of Formulae I-VII or any
combination thereof that increases the nuclease resistance of the siNA
construct.

[0171] In another embodiment, the invention features a method for
generating siNA molecules with increased nuclease resistance comprising
(a) introducing nucleotides having any of Formula I-VII or any
combination thereof into a siNA molecule, and (b) assaying the siNA
molecule of step (a) under conditions suitable for isolating siNA
molecules having increased nuclease resistance.

[0172] In one embodiment, the invention features siNA constructs that
mediate RNAi against a target gene, wherein the siNA construct comprises
one or more chemical modifications described herein that modulates the
binding affinity between the sense and antisense strands of the siNA
construct.

[0173] In one embodiment, the binding affinity between the sense and
antisense strands of the siNA construct is modulated to increase the
activity of the siNA molecule with regard to the ability of the siNA to
mediate RNA interference. In another embodiment the binding affinity
between the sense and antisense strands of the siNA construct is
decreased. The binding affinity between the sense and antisense strands
of the siNA construct can be decreased by introducing one or more
chemically modified nucleotides in the siNA sequence that disrupts the
duplex stability of the siNA (e.g., lowers the Tm of the duplex). The
binding affinity between the sense and antisense strands of the siNA
construct can be decreased by introducing one or more nucleotides in the
siNA sequence that do not form Watson-Crick base pairs. The binding
affinity between the sense and antisense strands of the siNA construct
can be decreased by introducing one or more wobble base pairs in the siNA
sequence. The binding affinity between the sense and antisense strands of
the siNA construct can be decreased by modifying the nucleobase
composition of the siNA, such as by altering the G-C content of the siNA
sequence (e.g., decreasing the number of G-C base pairs in the siNA
sequence). These modifications and alterations in sequence can be
introduced selectively at pre-determined positions of the siNA sequence
to increase siNA mediated RNAi activity. For example, such modifications
and sequence alterations can be introduced to disrupt siNA duplex
stability between the 5'-end of the antisense strand and the 3'-end of
the sense strand, the 3'-end of the antisense strand and the 5'-end of
the sense strand, or alternately the middle of the siNA duplex. In
another embodiment, siNA molecules are screened for optimized RNAi
activity by introducing such modifications and sequence alterations
either by rational design based upon observed rules or trends in
increasing siNA activity, or randomly via combinatorial selection
processes that cover either partial or complete sequence space of the
siNA construct.

[0174] In another embodiment, the invention features a method for
generating siNA molecules with increased binding affinity between the
sense and antisense strands of the siNA molecule comprising (a)
introducing nucleotides having any of Formula I-VII or any combination
thereof into a siNA molecule, and (b) assaying the siNA molecule of step
(a) under conditions suitable for isolating siNA molecules having
increased binding affinity between the sense and antisense strands of the
siNA molecule.

[0175] In one embodiment, the invention features siNA constructs that
mediate RNAi in a cell or reconstituted system, wherein the siNA
construct comprises one or more chemical modifications described herein
that modulates the binding affinity between the antisense strand of the
siNA construct and a complementary target RNA sequence within a cell.

[0176] In one embodiment, the invention features siNA constructs that
mediate RNAi in a cell or reconstituted system, wherein the siNA
construct comprises one or more chemical modifications described herein
that modulates the binding affinity between the antisense strand of the
siNA construct and a complementary target DNA sequence within a cell.

[0177] In another embodiment, the invention features a method for
generating siNA molecules with increased binding affinity between the
antisense strand of the siNA molecule and a complementary target RNA
sequence comprising (a) introducing nucleotides having any of Formula
I-VII or any combination thereof into a siNA molecule, and (b) assaying
the siNA molecule of step (a) under conditions suitable for isolating
siNA molecules having increased binding affinity between the antisense
strand of the siNA molecule and a complementary target RNA sequence.

[0178] In another embodiment, the invention features a method for
generating siNA molecules with increased binding affinity between the
antisense strand of the siNA molecule and a complementary target DNA
sequence comprising (a) introducing nucleotides having any of Formula
I-VII or any combination thereof into a siNA molecule, and (b) assaying
the siNA molecule of step (a) under conditions suitable for isolating
siNA molecules having increased binding affinity between the antisense
strand of the siNA molecule and a complementary target DNA sequence.

[0179] In one embodiment, the invention features siNA constructs that
mediate RNAi in a cell or reconstituted system, wherein the siNA
construct comprises one or more chemical modifications described herein
that modulate the polymerase activity of a cellular polymerase capable of
generating additional endogenous siNA molecules having sequence homology
to the chemically-modified siNA construct.

[0180] In another embodiment, the invention features a method for
generating siNA molecules capable of mediating increased polymerase
activity of a cellular polymerase capable of generating additional
endogenous siNA molecules having sequence homology to a
chemically-modified siNA molecule comprising (a) introducing nucleotides
having any of Formula I-VII or any combination thereof into a siNA
molecule, and (b) assaying the siNA molecule of step (a) under conditions
suitable for isolating siNA molecules capable of mediating increased
polymerase activity of a cellular polymerase capable of generating
additional endogenous siNA molecules having sequence homology to the
chemically-modified siNA molecule. In one embodiment, the invention
features chemically-modified siNA constructs that mediate RNAi in a cell
or reconstituted system, wherein the chemical modifications do not
significantly effect the interaction of siNA with a target RNA molecule,
DNA molecule and/or proteins or other factors that are essential for RNAi
in a manner that would decrease the efficacy of RNAi mediated by such
siNA constructs.

[0181] In another embodiment, the invention features a method for
generating siNA molecules with improved RNAi activity comprising (a)
introducing nucleotides having any of Formula I-VII or any combination
thereof into a siNA molecule, and (b) assaying the siNA molecule of step
(a) under conditions suitable for isolating siNA molecules having
improved RNAi activity.

[0182] In yet another embodiment, the invention features a method for
generating siNA molecules with improved RNAi activity against a target
RNA comprising (a) introducing nucleotides having any of Formula I-VII or
any combination thereof into a siNA molecule, and (b) assaying the siNA
molecule of step (a) under conditions suitable for isolating siNA
molecules having improved RNAi activity against the target RNA.

[0183] In yet another embodiment, the invention features a method for
generating siNA molecules with improved RNAi activity against a DNA
target comprising (a) introducing nucleotides having any of Formula I-VII
or any combination thereof into a siNA molecule, and (b) assaying the
siNA molecule of step (a) under conditions suitable for isolating siNA
molecules having improved RNAi activity against the DNA target, such as a
gene, chromosome, or portion thereof.

[0184] In one embodiment, the invention features siNA constructs that
mediate RNAi in a cell or reconstituted system, wherein the siNA
construct comprises one or more chemical modifications described herein
that modulates the cellular uptake of the siNA construct.

[0185] In another embodiment, the invention features a method for
generating siNA molecules against a target gene with improved cellular
uptake comprising (a) introducing nucleotides having any of Formula I-VII
or any combination thereof into a siNA molecule, and (b) assaying the
siNA molecule of step (a) under conditions suitable for isolating siNA
molecules having improved cellular uptake.

[0186] In one embodiment, the invention features siNA constructs that
mediate RNAi against a target gene, wherein the siNA construct comprises
one or more chemical modifications described herein that increases the
bioavailability of the siNA construct, for example, by attaching
polymeric conjugates such as polyethyleneglycol or equivalent conjugates
that improve the pharmacokinetics of the siNA construct, or by attaching
conjugates that target specific tissue types or cell types in vivo.
Non-limiting examples of such conjugates are described in Vargeese et
al., U.S. Ser. No. 10/201,394 incorporated by reference herein.

[0187] In one embodiment, the invention features a method for generating
siNA molecules of the invention with improved bioavailability comprising
(a) introducing a conjugate into the structure of a siNA molecule, and
(b) assaying the siNA molecule of step (a) under conditions suitable for
isolating siNA molecules having improved bioavailability. Such conjugates
can include ligands for cellular receptors, such as peptides derived from
naturally occurring protein ligands; protein localization sequences,
including cellular ZIP code sequences; antibodies; nucleic acid aptamers;
vitamins and other co-factors, such as folate and N-acetylgalactosamine;
polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;
polyamines, such as spermine or spermidine; and others.

[0188] In one embodiment, the invention features a double stranded short
interfering nucleic acid (siNA) molecule that comprises a first
nucleotide sequence complementary to a target RNA sequence or a portion
thereof, and a second sequence having complementarity to said first
sequence, wherein said second sequence is chemically modified in a manner
that it can no longer act as a guide sequence for efficiently mediating
RNA interference and/or is recognized by cellular proteins that
facilitate RNAi.

[0189] In one embodiment, the invention features a double stranded short
interfering nucleic acid (siNA) molecule that comprises a first
nucleotide sequence complementary to a target RNA sequence or a portion
thereof, and a second sequence having complementarity to said first
sequence, wherein the second sequence is designed or modified in a manner
that prevents its entry into the RNAi pathway as a guide sequence or as a
sequence that is complementary to a target nucleic acid (e.g., RNA)
sequence. Such design or modifications are expected to enhance the
activity of siNA and/or improve the specificity of siNA molecules of the
invention. These modifications are also expected to minimize any
off-target effects and/or associated toxicity.

[0190] In one embodiment, the invention features a double stranded short
interfering nucleic acid (siNA) molecule that comprises a first
nucleotide sequence complementary to a target RNA sequence or a portion
thereof, and a second sequence having complementarity to said first
sequence, wherein said second sequence is incapable of acting as a guide
sequence for mediating RNA interference.

[0191] In one embodiment, the invention features a double stranded short
interfering nucleic acid (siNA) molecule that comprises a first
nucleotide sequence complementary to a target RNA sequence or a portion
thereof, and a second sequence having complementarity to said first
sequence, wherein said second sequence does not have a terminal
5'-hydroxyl (5'-OH) or 5'-phosphate group.

[0192] In one embodiment, the invention features a double stranded short
interfering nucleic acid (siNA) molecule that comprises a first
nucleotide sequence complementary to a target RNA sequence or a portion
thereof, and a second sequence having complementarity to said first
sequence, wherein said second sequence comprises a terminal cap moiety at
the 5'-end of said second sequence. In another embodiment, the terminal
cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted
nucleotide moiety, a group shown in FIG. 22, an alkyl or cycloalkyl
group, a heterocycle, or any other group that prevents RNAi activity in
which the second sequence serves as a guide sequence or template for
RNAi.

[0193] In one embodiment, the invention features a double stranded short
interfering nucleic acid (siNA) molecule that comprises a first
nucleotide sequence complementary to a target RNA sequence or a portion
thereof, and a second sequence having complementarity to said first
sequence, wherein said second sequence comprises a terminal cap moiety at
the 5'-end and 3'-end of said second sequence. In another embodiment,
each terminal cap moiety individually comprises an inverted abasic,
inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.
22, an alkyl or cycloalkyl group, a heterocycle, or any other group that
prevents RNAi activity in which the second sequence serves as a guide
sequence or template for RNAi.

[0194] In one embodiment, the invention features a method for generating
siNA molecules of the invention with improved specificity for down
regulating or inhibiting the expression of a target nucleic acid (e.g., a
DNA or RNA such as a gene or its corresponding RNA), comprising (a)
introducing one or more chemical modifications into the structure of a
siNA molecule, and (b) assaying the siNA molecule of step (a) under
conditions suitable for isolating siNA molecules having improved
specificity. In another embodiment, the chemical modification used to
improve specificity comprises terminal cap modifications at the 5'-end,
3'-end, or both 5' and 3'-ends of the siNA molecule. The terminal cap
modifications can comprise, for example, structures shown in FIG. 22
(e.g. inverted deoxyabasic moieties) or any other chemical modification
that renders a portion of the siNA molecule (e.g. the sense strand)
incapable of mediating RNA interference against an off target nucleic
acid sequence. In a non-limiting example, a siNA molecule is designed
such that only the antisense sequence of the siNA molecule can serve as a
guide sequence for RISC mediated degradation of a corresponding target
RNA sequence. This can be accomplished by rendering the sense sequence of
the siNA inactive by introducing chemical modifications to the sense
strand that preclude recognition of the sense strand as a guide sequence
by RNAi machinery. In one embodiment, such chemical modifications
comprise any chemical group at the 5'-end of the sense strand of the
siNA, or any other group that serves to render the sense strand inactive
as a guide sequence for mediating RNA interference. These modifications,
for example, can result in a molecule where the 5'-end of the sense
strand no longer has a free 5'-hydroxyl (5'-OH) or a free 5'-phosphate
group (e.g., phosphate, diphosphate, triphosphate, cyclic phosphate
etc.). Non-limiting examples of such siNA constructs are described
herein, such as "Stab 9/10" and "Stab 7/8" chemistries and variants
thereof wherein the 5'-end and 3'-end of the sense strand of the siNA do
not comprise a hydroxyl group or phosphate group.

[0195] In one embodiment, the invention features a method for generating
siNA molecules of the invention with improved specificity for down
regulating or inhibiting the expression of a target nucleic acid (e.g., a
DNA or RNA such as a gene or its corresponding RNA), comprising (a)
introducing one or more chemical modifications into the structure of a
siNA molecule that prevent a strand or portion of the siNA molecule from
acting as a template or guide sequence for RNAi activity. In another
embodiment, the inactive strand or sense region of the siNA molecule is
the sense strand or sense region of the siNA molecule, i.e. the strand or
region of the siNA that does not have complementarity to the target
nucleic acid sequence. In one embodiment, such chemical modifications
comprise any chemical group at the 5'-end of the sense strand or region
of the siNA that does not comprise a 5'-hydroxyl (5'-OH) or 5'-phosphate
group, or any other group that serves to render the sense strand or sense
region inactive as a guide sequence for mediating RNA interference.
Non-limiting examples of such siNA constructs are described herein, such
as "Stab 9/10" and "Stab 7/8" chemistries and variants thereof wherein
the 5'-end and 3'-end of the sense strand of the siNA do not comprise a
hydroxyl group or phosphate group.

[0196] In one embodiment, the invention features a method for screening
siNA molecules against a target nucleic acid sequence comprising, (a)
generating a plurality of unmodified siNA molecules, (b) assaying the
siNA molecules of step (a) under conditions suitable for isolating siNA
molecules that are active in mediating RNA interference against the
target nucleic acid sequence, (c) introducing chemical modifications
(e.g. chemical modifications as described herein or as otherwise known in
the art) into the active siNA molecules of (b), and (d) optionally
re-screening the chemically modified siNA molecules of (c) under
conditions suitable for isolating chemically modified siNA molecules that
are active in mediating RNA interference against the target nucleic acid
sequence.

[0197] In one embodiment, the invention features a method for screening
siNA molecules against a target nucleic acid sequence comprising, (a)
generating a plurality of chemically modified siNA molecules (e.g. siNA
molecules as described herein or as otherwise known in the art), and (b)
assaying the siNA molecules of step (a) under conditions suitable for
isolating chemically modified siNA molecules that are active in mediating
RNA interference against the target nucleic acid sequence.

[0198] In another embodiment, the invention features a method for
generating siNA molecules of the invention with improved bioavailability
comprising (a) introducing an excipient formulation to a siNA molecule,
and (b) assaying the siNA molecule of step (a) under conditions suitable
for isolating siNA molecules having improved bioavailability. Such
excipients include polymers such as cyclodextrins, lipids, cationic
lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and
others.

[0199] In another embodiment, the invention features a method for
generating siNA molecules of the invention with improved bioavailability
comprising (a) introducing an excipient formulation to a siNA molecule,
and (b) assaying the siNA molecule of step (a) under conditions suitable
for isolating siNA molecules having improved bioavailability. Such
excipients include polymers such as cyclodextrins, lipids, cationic
lipids, polyamines, phospholipids, and others.

[0200] In another embodiment, the invention features a method for
generating siNA molecules of the invention with improved bioavailability
comprising (a) introducing nucleotides having any of Formulae I-VII or
any combination thereof into a siNA molecule, and (b) assaying the siNA
molecule of step (a) under conditions suitable for isolating siNA
molecules having improved bioavailability.

[0201] In another embodiment, polyethylene glycol (PEG) can be covalently
attached to siNA compounds of the present invention. The attached PEG can
be any molecular weight, preferably from about 2,000 to about 50,000
daltons (Da).

[0202] The present invention can be used alone or as a component of a kit
having at least one of the reagents necessary to carry out the in vitro
or in vivo introduction of RNA to test samples and/or subjects. For
example, preferred components of the kit include a siNA molecule of the
invention and a vehicle that promotes introduction of the siNA into cells
of interest as described herein (e.g., using lipids and other methods of
transfection known in the art, see for example Beigelman et al, U.S. Pat.
No. 6,395,713). The kit can be used for target validation, such as in
determining gene function and/or activity, or in drug optimization, and
in drug discovery (see for example Usman et al., U.S. Ser. No.
60/402,996). Such a kit can also include instructions to allow a user of
the kit to practice the invention.

[0203] The term "short interfering nucleic acid", "siNA", "short
interfering RNA", "siRNA", "short interfering nucleic acid molecule",
"short interfering oligonucleotide molecule", or "chemically-modified
short interfering nucleic acid molecule" as used herein refers to any
nucleic acid molecule capable of inhibiting or down regulating gene
expression or viral replication, for example by mediating RNA
interference "RNAi" or gene silencing in a sequence-specific manner; see
for example Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature,
411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer
et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et
al., International PCT Publication No. WO 01/36646; Fire, International
PCT Publication No. WO 99/32619; Plaetinck et al., International PCT
Publication No. WO 00/01846; Mello and Fire, International PCT
Publication No. WO 01/29058; Deschamps-Depaillette, International PCT
Publication No. WO 99/07409; and Li et al., International PCT Publication
No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,
2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218;
and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore,
2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850;
Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel,
2002, Science, 297, 1831). Non limiting examples of siNA molecules of the
invention are shown in FIGS. 18-20, and Table I herein. For example the
siNA can be a double-stranded polynucleotide molecule comprising
self-complementary sense and antisense regions, wherein the antisense
region comprises nucleotide sequence that is complementary to nucleotide
sequence in a target nucleic acid molecule or a portion thereof and the
sense region having nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof. The siNA can be assembled
from two separate oligonucleotides, where one strand is the sense strand
and the other is the antisense strand, wherein the antisense and sense
strands are self-complementary (i.e. each strand comprises nucleotide
sequence that is complementary to nucleotide sequence in the other
strand; such as where the antisense strand and sense strand form a duplex
or double stranded structure, for example wherein the double stranded
region is about 19 base pairs); the antisense strand comprises nucleotide
sequence that is complementary to nucleotide sequence in a target nucleic
acid molecule or a portion thereof and the sense strand comprises
nucleotide sequence corresponding to the target nucleic acid sequence or
a portion thereof. Alternatively, the siNA is assembled from a single
oligonucleotide, where the self-complementary sense and antisense regions
of the siNA are linked by means of a nucleic acid based or non-nucleic
acid-based linker(s). The siNA can be a polynucleotide with a duplex,
asymmetric duplex, hairpin or asymmetric hairpin secondary structure,
having self-complementary sense and antisense regions, wherein the
antisense region comprises nucleotide sequence that is complementary to
nucleotide sequence in a separate target nucleic acid molecule or a
portion thereof and the sense region having nucleotide sequence
corresponding to the target nucleic acid sequence or a portion thereof.
The siNA can be a circular single-stranded polynucleotide having two or
more loop structures and a stem comprising self-complementary sense and
antisense regions, wherein the antisense region comprises nucleotide
sequence that is complementary to nucleotide sequence in a target nucleic
acid molecule or a portion thereof and the sense region having nucleotide
sequence corresponding to the target nucleic acid sequence or a portion
thereof, and wherein the circular polynucleotide can be processed either
in vivo or in vitro to generate an active siNA molecule capable of
mediating RNAi. The siNA can also comprise a single stranded
polynucleotide having nucleotide sequence complementary to nucleotide
sequence in a target nucleic acid molecule or a portion thereof (for
example, where such siNA molecule does not require the presence within
the siNA molecule of nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof), wherein the single stranded
polynucleotide can further comprise a terminal phosphate group, such as a
5'-phosphate (see for example Martinez et al., 2002, Cell, 110, 563-574
and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or
5',3'-diphosphate. In certain embodiments, the siNA molecule of the
invention comprises separate sense and antisense sequences or regions,
wherein the sense and antisense regions are covalently linked by
nucleotide or non-nucleotide linkers molecules as is known in the art, or
are alternately non-covalently linked by ionic interactions, hydrogen
bonding, van der waals interactions, hydrophobic intercations, and/or
stacking interactions. In certain embodiments, the siNA molecules of the
invention comprise nucleotide sequence that is complementary to
nucleotide sequence of a target gene. In another embodiment, the siNA
molecule of the invention interacts with nucleotide sequence of a target
gene in a manner that causes inhibition of expression of the target gene.
As used herein, siNA molecules need not be limited to those molecules
containing only RNA, but further encompasses chemically-modified
nucleotides and non-nucleotides. In certain embodiments, the short
interfering nucleic acid molecules of the invention lack 2'-hydroxy
(2'-OH) containing nucleotides. Applicant describes in certain
embodiments short interfering nucleic acids that do not require the
presence of nucleotides having a 2'-hydroxy group for mediating RNAi and
as such, short interfering nucleic acid molecules of the invention
optionally do not include any ribonucleotides (e.g., nucleotides having a
2'-OH group). Such siNA molecules that do not require the presence of
ribonucleotides within the siNA molecule to support RNAi can however have
an attached linker or linkers or other attached or associated groups,
moieties, or chains containing one or more nucleotides with 2'-OH groups.
Optionally, siNA molecules can comprise ribonucleotides at about 5, 10,
20, 30, 40, or 50% of the nucleotide positions. The modified short
interfering nucleic acid molecules of the invention can also be referred
to as short interfering modified oligonucleotides "siMON." As used
herein, the term siNA is meant to be equivalent to other terms used to
describe nucleic acid molecules that are capable of mediating sequence
specific RNAi, for example short interfering RNA (siRNA), double-stranded
RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short
interfering oligonucleotide, short interfering nucleic acid, short
interfering modified oligonucleotide, chemically-modified siRNA,
post-transcriptional gene silencing RNA (ptgsRNA), and others. In
addition, as used herein, the term RNAi is meant to be equivalent to
other terms used to describe sequence specific RNA interference, such as
post transcriptional gene silencing, translational inhibition, or
epigenetics. For example, siNA molecules of the invention can be used to
epigenetically silence genes at both the post-transcriptional level or
the pre-transcriptional level. In a non-limiting example, epigenetic
regulation of gene expression by siNA molecules of the invention can
result from siNA mediated modification of chromatin structure to alter
gene expression (see, for example, Allshire, 2002, Science, 297,
1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002,
Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

[0204] By "asymmetric hairpin" as used herein is meant a linear siNA
molecule comprising an antisense region, a loop portion that can comprise
nucleotides or non-nucleotides, and a sense region that comprises fewer
nucleotides than the antisense region to the extent that the sense region
has enough complimentary nucleotides to base pair with the antisense
region and form a duplex with loop. For example, an asymmetric hairpin
siNA molecule of the invention can comprise an antisense region having
length sufficient to mediate RNAi in a cell or in vitro system (e.g.
about 19 to about 22 nucleotides) and a loop region comprising about 4 to
about 8 nucleotides, and a sense region having about 3 to about 18
nucleotides that are complementary to the antisense region (see for
example FIG. 74). The asymmetric hairpin siNA molecule can also comprise
a 5'-terminal phosphate group that can be chemically modified (for
example as shown in FIG. 75). The loop portion of the asymmetric hairpin
siNA molecule can comprise nucleotides, non-nucleotides, linker
molecules, or conjugate molecules as described herein.

[0205] By "asymmetric duplex" as used herein is meant a siNA molecule
having two separate strands comprising a sense region and an antisense
region, wherein the sense region comprises fewer nucleotides than the
antisense region to the extent that the sense region has enough
complimentary nucleotides to base pair with the antisense region and form
a duplex. For example, an asymmetric duplex siNA molecule of the
invention can comprise an antisense region having length sufficient to
mediate RNAi in a cell or in vitro system (e.g. about 19 to about 22
nucleotides) and a sense region having about 3 to about 18 nucleotides
that are complementary to the antisense region (see for example FIG. 74).

[0206] By "modulate" is meant that the expression of the gene, or level of
RNA molecule or equivalent RNA molecules encoding one or more proteins or
protein subunits, or activity of one or more proteins or protein subunits
is up regulated or down regulated, such that expression, level, or
activity is greater than or less than that observed in the absence of the
modulator. For example, the term "modulate" can mean "inhibit," but the
use of the word "modulate" is not limited to this definition.

[0207] By "inhibit", "down-regulate", or "reduce", it is meant that the
expression of the gene, or level of RNA molecules or equivalent RNA
molecules encoding one or more proteins or protein subunits, or activity
of one or more proteins or protein subunits, is reduced below that
observed in the absence of the nucleic acid molecules (e.g., siNA) of the
invention. In one embodiment, inhibition, down-regulation or reduction
with an siNA molecule is below that level observed in the presence of an
inactive or attenuated molecule. In another embodiment, inhibition,
down-regulation, or reduction with siNA molecules is below that level
observed in the presence of, for example, an siNA molecule with scrambled
sequence or with mismatches. In another embodiment, inhibition,
down-regulation, or reduction of gene expression with a nucleic acid
molecule of the instant invention is greater in the presence of the
nucleic acid molecule than in its absence.

[0208] By "gene", or "target gene", is meant, a nucleic acid that encodes
an RNA, for example, nucleic acid sequences including, but not limited
to, structural genes encoding a polypeptide. A gene or target gene can
also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as
small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),
short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA
(rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding
RNAs can serve as target nucleic acid molecules for siNA mediated RNA
interference in modulating the activity of fRNA or ncRNA involved in
functional or regulatory cellular processes. Abberant fRNA or ncRNA
activity leading to disease can therefore be modulated by siNA molecules
of the invention. siNA molecules targeting fRNA and ncRNA can also be
used to manipulate or alter the genotype or phenotype of an organism or
cell, by intervening in cellular processes such as genetic imprinting,
transcription, translation, or nucleic acid processing (e.g.,
transamination, methylation etc.). The target gene can be a gene derived
from a cell, an endogenous gene, a transgene, or exogenous genes such as
genes of a pathogen, for example a virus, which is present in the cell
after infection thereof. The cell containing the target gene can be
derived from or contained in any organism, for example a plant, animal,
protozoan, virus, bacterium, or fungus. Non-limiting examples of plants
include monocots, dicots, or gymnosperms. Non-limiting examples of
animals include vertebrates or invertebrates. Non-limiting examples of
fungi include molds or yeasts.

[0209] By "highly conserved sequence region" is meant, a nucleotide
sequence of one or more regions in a target gene does not vary
significantly from one generation to the other or from one biological
system to the other.

[0210] By "cancer" is meant a group of diseases characterized by
uncontrolled growth and spread of abnormal cells.

[0211] By "sense region" is meant a nucleotide sequence of a siNA molecule
having complementarity to an antisense region of the siNA molecule. In
addition, the sense region of a siNA molecule can comprise a nucleic acid
sequence having homology with a target nucleic acid sequence.

[0212] By "antisense region" is meant a nucleotide sequence of a siNA
molecule having complementarity to a target nucleic acid sequence. In
addition, the antisense region of a siNA molecule can optionally comprise
a nucleic acid sequence having complementarity to a sense region of the
siNA molecule.

[0213] By "target nucleic acid" is meant any nucleic acid sequence whose
expression or activity is to be modulated. The target nucleic acid can be
DNA or RNA, such as endogenous DNA or RNA, viral DNA or viral RNA, or
other RNA encoded by a gene, virus, bacteria, fungus, mammal, or plant.

[0214] By "complementarity" is meant that a nucleic acid can form hydrogen
bond(s) with another nucleic acid sequence by either traditional
Watson-Crick or other non-traditional types. In reference to the nucleic
molecules of the present invention, the binding free energy for a nucleic
acid molecule with its complementary sequence is sufficient to allow the
relevant function of the nucleic acid to proceed, e.g., RNAi activity.
Determination of binding free energies for nucleic acid molecules is well
known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol.
LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA
83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A
percent complementarity indicates the percentage of contiguous residues
in a nucleic acid molecule that can form hydrogen bonds (e.g.,
Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,
6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the
first oligonucleotide being based paired to a second nucleic acid
sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and
100% complementary respectively). "Perfectly complementary" means that
all the contiguous residues of a nucleic acid sequence will hydrogen bond
with the same number of contiguous residues in a second nucleic acid
sequence.

[0215] The siNA molecules of the invention represent a novel therapeutic
approach to a broad spectrum of diseases and conditions, including cancer
or cancerous disease, infectious disease, cardiovascular disease,
neurological disease, prion disease, inflammatory disease, autoimmune
disease, pulmonary disease, renal disease, liver disease, mitochondrial
disease, endocrine disease, reproduction related diseases and conditions,
and any other indications that can respond to the level of an expressed
gene product in a cell or organism.

[0216] In one embodiment of the present invention, each sequence of a siNA
molecule of the invention is independently about 18 to about 24
nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22,
23, or 24 nucleotides in length. In another embodiment, the siNA duplexes
of the invention independently comprise about 17 to about 23 base pairs
(e.g., about 17, 18, 19, 20, 21, 22 or 23). In yet another embodiment,
siNA molecules of the invention comprising hairpin or circular structures
are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides
in length, or about 38 to about 44 (e.g., 38, 39, 40, 41, 42, 43 or 44)
nucleotides in length and comprising about 16 to about 22 (e.g., about
16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNA molecules of the
invention are shown in Table I. and/or FIGS. 18-19.

[0217] As used herein "cell" is used in its usual biological sense, and
does not refer to an entire multicellular organism, e.g., specifically
does not refer to a human. The cell can be present in an organism, e.g.,
birds, plants and mammals such as humans, cows, sheep, apes, monkeys,
swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell)
or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic
or germ line origin, totipotent or pluripotent, dividing or non-dividing.
The cell can also be derived from or can comprise a gamete or embryo, a
stem cell, or a fully differentiated cell.

[0218] The siNA molecules of the invention are added directly, or can be
complexed with cationic lipids, packaged within liposomes, or otherwise
delivered to target cells or tissues. The nucleic acid or nucleic acid
complexes can be locally administered to relevant tissues ex vivo, or in
vivo through injection, infusion pump or stent, with or without their
incorporation in biopolymers. In particular embodiments, the nucleic acid
molecules of the invention comprise sequences shown in Table I and/or
FIGS. 18-19. Examples of such nucleic acid molecules consist essentially
of sequences defined in these tables and figures. Furthermore, the
chemically modified constructs described in Table IV can be applied to
any siNA sequence of the invention.

[0219] In another aspect, the invention provides mammalian cells
containing one or more siNA molecules of this invention. The one or more
siNA molecules can independently be targeted to the same or different
sites.

[0220] By "RNA" is meant a molecule comprising at least one ribonucleotide
residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group
at the 2' position of a 13-D-ribo-furanose moiety. The terms include
double-stranded RNA, single-stranded RNA, isolated RNA such as partially
purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced
RNA, as well as altered RNA that differs from naturally occurring RNA by
the addition, deletion, substitution and/or alteration of one or more
nucleotides. Such alterations can include addition of non-nucleotide
material, such as to the end(s) of the siNA or internally, for example at
one or more nucleotides of the RNA. Nucleotides in the RNA molecules of
the instant invention can also comprise non-standard nucleotides, such as
non-naturally occurring nucleotides or chemically synthesized nucleotides
or deoxynucleotides. These altered RNAs can be referred to as analogs or
analogs of naturally-occurring RNA.

[0221] By "subject" is meant an organism, which is a donor or recipient of
explanted cells or the cells themselves. "Subject" also refers to an
organism to which the nucleic acid molecules of the invention can be
administered. A subject can be a mammal or mammalian cells, including a
human or human cells.

[0222] The term "ligand" refers to any compound or molecule, such as a
drug, peptide, hormone, or neurotransmitter, that is capable of
interacting with another compound, such as a receptor, either directly or
indirectly. The receptor that interacts with a ligand can be present on
the surface of a cell or can alternately be an intercellular receptor.
Interaction of the ligand with the receptor can result in a biochemical
reaction, or can simply be a physical interaction or association.

[0223] The term "phosphorothioate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z and/or W comprise a
sulfur atom. Hence, the term phosphorothioate refers to both
phosphorothioate and phosphorodithioate internucleotide linkages.

[0224] The term "phosphonoacetate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z and/or W comprise an
acetyl or protected acetyl group.

[0225] The term "thiophosphonoacetate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z comprises an acetyl
or protected acetyl group and W comprises a sulfur atom or alternately W
comprises an acetyl or protected acetyl group and Z comprises a sulfur
atom.

[0226] The term "universal base" as used herein refers to nucleotide base
analogs that form base pairs with each of the natural DNA/RNA bases with
little discrimination between them. Non-limiting examples of universal
bases include C-phenyl, C-naphthyl and other aromatic derivatives,
inosine, azole carboxamides, and nitroazole derivatives such as
3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known
in the art (see for example Loakes, 2001, Nucleic Acids Research, 29,
2437-2447).

[0227] The term "acyclic nucleotide" as used herein refers to any
nucleotide having an acyclic ribose sugar, for example where any of the
ribose carbons (C1, C2, C3, C4, or C5), are independently or in
combination absent from the nucleotide.

[0228] The nucleic acid molecules of the instant invention, individually,
or in combination or in conjunction with other drugs, can be used to
treat diseases or conditions discussed herein (e.g., cancers and other
proliferative conditions, viral infection, inflammatory disease,
autoimmunity, pulmonary disease, renal disease, ocular disease, etc.).
For example, to treat a particular disease or condition, the siNA
molecules can be administered to a subject or can be administered to
other appropriate cells evident to those skilled in the art, individually
or in combination with one or more drugs under conditions suitable for
the treatment.

[0229] In one embodiment, the invention features a method for treating or
preventing a disease or condition in a subject, wherein the disease or
condition is related to angiogenesis or neovascularization, comprising
administering to the subject a siNA molecule of the invention under
conditions suitable for the treatment or prevention of the disease or
condition in the subject, alone or in conjunction with one or more other
therapeutic compounds. In another embodiment, the disease or condition
comprises tumor angiogenesis and cancer, including but not limited to
breast cancer, lung cancer (including non-small cell lung carcinoma),
prostate cancer, colorectal cancer, brain cancer, esophageal cancer,
bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer,
skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial
carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid
adenocarcinoma, ovarian cancer, melanoma, lymphoma, glioma, endometrial
sarcoma, multidrug resistant cancers, diabetic retinopathy, macular
degeneration, age related macular degeneration, neovascular glaucoma,
myopic degeneration, arthritis, psoriasis, endometriosis, female
reproduction, verruca vulgaris, angiofibroma of tuberous sclerosis,
pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome,
Osler-Weber-Rendu syndrome, renal disease such as Autosomal dominant
polycystic kidney disease (ADPKD), restenosis, arteriosclerosis, and any
other diseases or conditions that are related to gene expression or will
respond to RNA interference in a cell or tissue, alone or in combination
with other therapies.

[0230] In one embodiment, the invention features a method for treating or
preventing an ocular disease or condition in a subject, wherein the
ocular disease or condition is related to angiogenesis or
neovascularization, comprising administering to the subject a siNA
molecule of the invention under conditions suitable for the treatment or
prevention of the disease or condition in the subject, alone or in
conjunction with one or more other therapeutic compounds. In another
embodiment, the ocular disease or condition comprises macular
degeneration, age related macular degeneration, diabetic retinopathy,
neovascular glaucoma, myopic degeneration, trachoma, scarring of the eye,
cataract, ocular inflammation and/or ocular infections.

[0231] In one embodiment, the invention features a method for treating or
preventing tumor angiogenesis in a subject, comprising administering to
the subject a siNA molecule of the invention under conditions suitable
for the treatment or prevention of tumor angiogenesis in the subject,
alone or in conjunction with one or more other therapeutic compounds.

[0232] In one embodiment, the invention features a method for treating or
preventing viral infection or replication in a subject, comprising
administering to the subject a siNA molecule of the invention under
conditions suitable for the treatment or prevention of viral infection or
replication in the subject, alone or in conjunction with one or more
other therapeutic compounds.

[0233] In one embodiment, the invention features a method for treating or
preventing autoimmune disease in a subject, comprising administering to
the subject a siNA molecule of the invention under conditions suitable
for the treatment or prevention of autoimmune disease in the subject,
alone or in conjunction with one or more other therapeutic compounds.

[0234] In one embodiment, the invention features a method for treating or
preventing inflammation in a subject, comprising administering to the
subject a siNA molecule of the invention under conditions suitable for
the treatment or prevention of inflammation in the subject, alone or in
conjunction with one or more other therapeutic compounds.

[0235] In a further embodiment, the siNA molecules can be used in
combination with other known treatments to treat conditions or diseases
discussed above. For example, the described molecules could be used in
combination with one or more known therapeutic agents to treat a disease
or condition. Non-limiting examples of other therapeutic agents that can
be readily combined with a siNA molecule of the invention are enzymatic
nucleic acid molecules, allosteric nucleic acid molecules, antisense,
decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal
antibodies, small molecules, and other organic and/or inorganic compounds
including metals, salts and ions.

[0236] Other features and advantages of the invention will be apparent
from the following description of the preferred embodiments thereof, and
from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0237] FIG. 1 shows a non-limiting example of a scheme for the synthesis
of siNA molecules. The complementary siNA sequence strands, strand 1 and
strand 2, are synthesized in tandem and are connected by a cleavable
linkage, such as a nucleotide succinate or abasic succinate, which can be
the same or different from the cleavable linker used for solid phase
synthesis on a solid support. The synthesis can be either solid phase or
solution phase, in the example shown, the synthesis is a solid phase
synthesis. The synthesis is performed such that a protecting group, such
as a dimethoxytrityl group, remains intact on the terminal nucleotide of
the tandem oligonucleotide. Upon cleavage and deprotection of the
oligonucleotide, the two siNA strands spontaneously hybridize to form a
siNA duplex, which allows the purification of the duplex by utilizing the
properties of the terminal protecting group, for example by applying a
trityl on purification method wherein only duplexes/oligonucleotides with
the terminal protecting group are isolated.

[0238] FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex
synthesized by a method of the invention. The two peaks shown correspond
to the predicted mass of the separate siNA sequence strands. This result
demonstrates that the siNA duplex generated from tandem synthesis can be
purified as a single entity using a simple trityl-on purification
methodology.

[0239]FIG. 3 shows the results of a stability assay used to determine the
serum stability of chemically modified siNA constructs compared to a siNA
control consisting of all RNA with 3'-TT termini. T 1/2 values are shown
for duplex stability.

[0240] FIG. 4 shows the results of an RNAi activity screen of several
phosphorothioate modified siNA constructs using a luciferase reporter
system.

[0241] FIG. 5 shows the results of an RNAi activity screen of several
phosphorothioate and universal base modified siNA constructs using a
luciferase reporter system.

[0242] FIG. 6 shows the results of an RNAi activity screen of several
2'-O-methyl modified siNA constructs using a luciferase reporter system.

[0243] FIG. 7 shows the results of an RNAi activity screen of several
2'-O-methyl and 2'-deoxy-2'-fluoro modified siNA constructs using a
luciferase reporter system.

[0244] FIG. 8 shows the results of an RNAi activity screen of a
phosphorothioate modified siNA construct using a luciferase reporter
system.

[0246] FIG. 10 shows the results of an RNAi activity screen of chemically
modified siNA constructs including 3'-glyceryl modified siNA constructs
compared to an all RNA control siNA construct using a luciferase reporter
system. These chemically modified siNAs were compared in the luciferase
assay described herein at 1 nM and 10 nM concentration using an all RNA
siNA control (siGL2) having 3'-terminal dithymidine (TT) and its
corresponding inverted control (Inv siGL2). The background level of
luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.

[0247] FIG. 11 shows the results of an RNAi activity screen of chemically
modified siNA constructs. The screen compared various combinations of
sense strand chemical modifications and antisense strand chemical
modifications. These chemically modified siNAs were compared in the
luciferase assay described herein at 1 nM and 10 nM concentration using
an all RNA siNA control (siGL2) having 3'-terminal dithymidine (TT) and
its corresponding inverted control (Inv siGL2). The background level of
luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.

[0248] FIG. 12 shows the results of an RNAi activity screen of chemically
modified siNA constructs. The screen compared various combinations of
sense strand chemical modifications and antisense strand chemical
modifications. These chemically modified siNAs were compared in the
luciferase assay described herein at 1 nM and 10 nM concentration using
an all RNA siNA control (siGL2) having 3'-terminal dithymidine (TT) and
its corresponding inverted control (Inv siGL2). The background level of
luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.
In addition, the antisense strand alone (Sirna/RPI 30430) and an inverted
control (Sirna/RPI 30227/30229, having matched chemistry to Sirna/RPI
(30063/30224) was compared to the siNA duplexes described above.

[0249] FIG. 13 shows the results of an RNAi activity screen of chemically
modified siNA constructs. The screen compared various combinations of
sense strand chemical modifications and antisense strand chemical
modifications. These chemically modified siNAs were compared in the
luciferase assay described herein at 1 nM and 10 nM concentration using
an all RNA siNA control (siGL2) having 3'-terminal dithymidine (TT) and
its corresponding inverted control (Inv siGL2). The background level of
luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.
In addition, an inverted control (Sirna/RPI 30226/30229), having matched
chemistry to Sirna/RPI (30222/30224) was compared to the siNA duplexes
described above.

[0250] FIG. 14 shows the results of an RNAi activity screen of chemically
modified siNA constructs including various 3'-terminal modified siNA
constructs compared to an all RNA control siNA construct using a
luciferase reporter system. These chemically modified siNAs were compared
in the luciferase assay described herein at 1 nM and 10 nM concentration
using an all RNA siNA control (siGL2) having 3'-terminal dithymidine (TT)
and its corresponding inverted control (Inv siGL2). The background level
of luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.

[0251]FIG. 15 shows the results of an RNAi activity screen of chemically
modified siNA constructs. The screen compared various combinations of
sense strand chemistries compared to a fixed antisense strand chemistry.
These chemically modified siNAs were compared in the luciferase assay
described herein at 1 nM and 10 nM concentration using an all RNA siNA
control (siGL2) having 3'-terminal dithymidine (TT) and its corresponding
inverted control (Inv siGL2). The background level of luciferase
expression in the HeLa cells is designated by the "cells" column. Sense
and antisense strands of chemically modified siNA constructs are shown by
Sirna/RPI number (sense strand/antisense strand). Sequences corresponding
to these Sirna/RPI numbers are shown in Table I.

[0252] FIG. 16 shows the results of a siNA titration study using a
luciferase reporter system, wherein the RNAi activity of a
phosphorothioate modified siNA construct is compared to that of a siNA
construct consisting of all ribonucleotides except for two terminal
thymidine residues.

[0253] FIG. 17 shows a non-limiting proposed mechanistic representation of
target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),
which is generated by RNA-dependent RNA polymerase (RdRP) from foreign
single-stranded RNA, for example viral, transposon, or other exogenous
RNA, activates the DICER enzyme that in turn generates siNA duplexes.
Alternately, synthetic or expressed siNA can be introduced directly into
a cell by appropriate means. An active siNA complex forms which
recognizes a target RNA, resulting in degradation of the target RNA by
the RISC endonuclease complex or in the synthesis of additional RNA by
RNA-dependent RNA polymerase (RdRP), which can activate DICER and result
in additional siNA molecules, thereby amplifying the RNAi response.

[0254] FIG. 18A-F shows non-limiting examples of chemically-modified siNA
constructs of the present invention. In the figure, N stands for any
nucleotide (adenosine, guanosine, cytosine, uridine, or optionally
thymidine, for example thymidine can be substituted in the overhanging
regions designated by parenthesis (N N). Various modifications are shown
for the sense and antisense strands of the siNA constructs.

[0255] FIG. 18A: The sense strand comprises 21 nucleotides wherein the two
terminal 3'-nucleotides are optionally base paired and wherein all
nucleotides present are ribonucleotides except for (N N) nucleotides,
which can comprise ribonucleotides, deoxynucleotides, universal bases, or
other chemical modifications described herein. The antisense strand
comprises 21 nucleotides, optionally having a 3'-terminal glyceryl moiety
and wherein the two terminal 3'-nucleotides are optionally complementary
to the target RNA sequence, and wherein all nucleotides present are
ribonucleotides except for (N N) nucleotides, which can comprise
ribonucleotides, deoxynucleotides, universal bases, or other chemical
modifications described herein. A modified internucleotide linkage, such
as a phosphorothioate, phosphorodithioate or other modified
internucleotide linkage as described herein, shown as "s" connects the (N
N) nucleotides in the antisense strand.

[0256] FIG. 18B: The sense strand comprises 21 nucleotides wherein the two
terminal 3'-nucleotides are optionally base paired and wherein all
pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified nucleotides and all purine nucleotides that may be present are
2'-O-methyl modified nucleotides except for (N N) nucleotides, which can
comprise ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. The antisense strand comprises
21 nucleotides, optionally having a 3'-terminal glyceryl moiety and
wherein the two terminal 3'-nucleotides are optionally complementary to
the target RNA sequence, and wherein all pyrimidine nucleotides that may
be present are 2'-deoxy-2'-fluoro modified nucleotides and all purine
nucleotides that may be present are 2'-O-methyl modified nucleotides
except for (N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications
described herein. A modified internucleotide linkage, such as a
phosphorothioate, phosphorodithioate or other modified internucleotide
linkage as described herein, shown as "s" connects the (N N) nucleotides
in the sense and antisense strand.

[0257] FIG. 18C: The sense strand comprises 21 nucleotides having 5'- and
3'-terminal cap moieties wherein the two terminal 3'-nucleotides are
optionally base paired and wherein all pyrimidine nucleotides that may be
present are 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides except
for (N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications
described herein. The antisense strand comprises 21 nucleotides,
optionally having a 3'-terminal glyceryl moiety and wherein the two
terminal 3'-nucleotides are optionally complementary to the target RNA
sequence, and wherein all pyrimidine nucleotides that may be present are
2'-deoxy-2'-fluoro modified nucleotides except for (N N) nucleotides,
which can comprise ribonucleotides, deoxynucleotides, universal bases, or
other chemical modifications described herein. A modified internucleotide
linkage, such as a phosphorothioate, phosphorodithioate or other modified
internucleotide linkage as described herein, shown as "s" connects the (N
N) nucleotides in the antisense strand.

[0258] FIG. 18D: The sense strand comprises 21 nucleotides having 5'- and
3'-terminal cap moieties wherein the two terminal 3'-nucleotides are
optionally base paired and wherein all pyrimidine nucleotides that may be
present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N)
nucleotides, which can comprise ribonucleotides, deoxynucleotides,
universal bases, or other chemical modifications described herein and
wherein and all purine nucleotides that may be present are 2'-deoxy
nucleotides. The antisense strand comprises 21 nucleotides, optionally
having a 3'-terminal glyceryl moiety and wherein the two terminal
3'-nucleotides are optionally complementary to the target RNA sequence,
and wherein all pyrimidine nucleotides that may be present are
2'-deoxy-2'-fluoro modified nucleotides and all purine nucleotides that
may be present are 2'-β-methyl modified nucleotides except for (N N)
nucleotides, which can comprise ribonucleotides, deoxynucleotides,
universal bases, or other chemical modifications described herein. A
modified internucleotide linkage, such as a phosphorothioate,
phosphorodithioate or other modified internucleotide linkage as described
herein, shown as "s" connects the (N N) nucleotides in the antisense
strand.

[0259] FIG. 18E: The sense strand comprises 21 nucleotides having 5'- and
3'-terminal cap moieties wherein the two terminal 3'-nucleotides are
optionally base paired and wherein all pyrimidine nucleotides that may be
present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N)
nucleotides, which can comprise ribonucleotides, deoxynucleotides,
universal bases, or other chemical modifications described herein. The
antisense strand comprises 21 nucleotides, optionally having a
3'-terminal glyceryl moiety and wherein the two terminal 3'-nucleotides
are optionally complementary to the target RNA sequence, and wherein all
pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified nucleotides and all purine nucleotides that may be present are
2'-O-methyl modified nucleotides except for (N N) nucleotides, which can
comprise ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. A modified internucleotide
linkage, such as a phosphorothioate, phosphorodithioate or other modified
internucleotide linkage as described herein, shown as "s" connects the (N
N) nucleotides in the antisense strand.

[0260] FIG. 18F: The sense strand comprises 21 nucleotides having 5'- and
3'-terminal cap moieties wherein the two terminal 3'-nucleotides are
optionally base paired and wherein all pyrimidine nucleotides that may be
present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N)
nucleotides, which can comprise ribonucleotides, deoxynucleotides,
universal bases, or other chemical modifications described herein and
wherein and all purine nucleotides that may be present are 2'-deoxy
nucleotides. The antisense strand comprises 21 nucleotides, optionally
having a 3'-terminal glyceryl moiety and wherein the two terminal
3'-nucleotides are optionally complementary to the target RNA sequence,
and wherein all pyrimidine nucleotides that may be present are
2'-deoxy-2'-fluoro modified nucleotides and all purine nucleotides that
may be present are 2'-deoxy nucleotides except for (N N) nucleotides,
which can comprise ribonucleotides, deoxynucleotides, universal bases, or
other chemical modifications described herein. A modified internucleotide
linkage, such as a phosphorothioate, phosphorodithioate or other modified
internucleotide linkage as described herein, shown as "s" connects the (N
N) nucleotides in the antisense strand. The antisense strand of
constructs A-F comprise sequence complementary to any target nucleic acid
sequence of the invention. Furthermore, when a glyceryl moiety (L) is
present at the 3'-end of the antisense strand for any construct shown in
FIG. 4 A-F, the modified internucleotide linkage is optional.

[0262] FIG. 20 shows non-limiting examples of different siNA constructs of
the invention. The examples shown (constructs 1, 2, and 3) have 19
representative base pairs; however, different embodiments of the
invention include any number of base pairs described herein. Bracketed
regions represent nucleotide overhangs, for example comprising about 1,
2, 3, or 4 nucleotides in length when present, preferably about 2
nucleotides. Such overhangs can be present or absent (i.e., blunt ends).
Such blunt ends can be present on one end or both ends of the siNA
molecule, for example where all nucleotides present in a siNA duplex are
base paired. Constructs 1 and 2 can be used independently for RNAi
activity. Construct 2 can comprise a polynucleotide or non-nucleotide
linker, which can optionally be designed as a biodegradable linker. In
one embodiment, the loop structure shown in construct 2 can comprise a
biodegradable linker that results in the formation of construct 1 in vivo
and/or in vitro. In another example, construct 3 can be used to generate
construct 2 under the same principle wherein a linker is used to generate
the active siNA construct 2 in vivo and/or in vitro, which can optionally
utilize another biodegradable linker to generate the active siNA
construct 1 in vivo and/or in vitro. As such, the stability and/or
activity of the siNA constructs can be modulated based on the design of
the siNA construct for use in vivo or in vitro and/or in vitro.

[0263] FIG. 21 is a diagrammatic representation of a method used to
determine target sites for siNA mediated RNAi within a particular target
nucleic acid sequence, such as messenger RNA. (A) A pool of siNA
oligonucleotides are synthesized wherein the antisense region of the siNA
constructs has complementarity to target sites across the target nucleic
acid sequence, and wherein the sense region comprises sequence
complementary to the antisense region of the siNA. (B) The sequences are
transfected into cells. (C) Cells are selected based on phenotypic change
that is associated with modulation of the target nucleic acid sequence.
(D) The siNA is isolated from the selected cells and is sequenced to
identify efficacious target sites within the target nucleic acid
sequence.

[0264]FIG. 22 shows non-limiting examples of different stabilization
chemistries (1-10) that can be used, for example, to stabilize the 3'-end
of siNA sequences of the invention, including (1) [3-3']-inverted
deoxyribose; (2) deoxyribonucleotide; (3) [5'-3']-3'-deoxyribonucleotide;
(4) [5'-3']-ribonucleotide; (5) [5'-3']-3'-O-methyl ribonucleotide; (6)
3'-glyceryl; (7) [3'-5']-3'-deoxyribonucleotide; (8)
[3'-3']-deoxyribonucleotide; (9) [5'-2']-deoxyribonucleotide; and (10)
[5-3']-dideoxyribonucleotide. In addition to modified and unmodified
backbone chemistries indicated in the figure, these chemistries can be
combined with different backbone modifications as described herein, for
example, backbone modifications having Formula I. In addition, the
2'-deoxy nucleotide shown 5' to the terminal modifications shown can be
another modified or unmodified nucleotide or non-nucleotide described
herein, for example modifications having any of Formulae I-VII or any
combination thereof.

[0265] FIG. 23 shows a non-limiting example of siNA mediated inhibition of
VEGF-induced angiogenesis using the rat corneal model of angiogenesis.
siNA targeting site 2340 of VEGFR1 RNA (shown as Sirna/RPI No.
29695/29699) were compared to inverted controls (shown as Sirna/RPI No.
29983/29984) at three different concentrations and compared to a VEGF
control in which no siNA was administered.

[0267] FIG. 25 is a non-limiting example of a dose response HBsAg screen
of stabilized siNA constructs ("stab 4/5", see Table IV) targeting sites
262 and 1580 of the HBV pregenomic RNA in HepG2 cells at 0.5, 5, 10 and
25 nM compared to untreated and matched chemistry inverted sequence
controls. The siNA sense and antisense strands are shown by Sirna/RPI
number (sense/antisense).

[0268] FIG. 26 shows a dose response comparison of two different
stabilization chemistries ("stab 7/8" and "stab 7/11", see Table IV)
targeting site 1580 of the HBV pregenomic RNA in HepG2 cells at 5, 10,
25, 50 and 100 nM compared to untreated and matched chemistry inverted
sequence controls. The siNA sense and antisense strands are shown by
Sirna/RPI number (sense/antisense).

[0269] FIG. 27 shows a non-limiting example of a strategy used to identify
chemically modified siNA constructs of the invention that are nuclease
resistance while preserving the ability to mediate RNAi activity.
Chemical modifications are introduced into the siNA construct based on
educated design parameters (e.g. introducing 2'-modifications, base
modifications, backbone modifications, terminal cap modifications etc).
The modified construct in tested in an appropriate system (e.g human
serum for nuclease resistance, shown, or an animal model for PK/delivery
parameters). In parallel, the siNA construct is tested for RNAi activity,
for example in a cell culture system such as a luciferase reporter
assay). Lead siNA constructs are then identified which possess a
particular characteristic while maintaining RNAi activity, and can be
further modified and assayed once again. This same approach can be used
to identify siNA-conjugate molecules with improved pharmacokinetic
profiles, delivery, and RNAi activity.

[0270] FIG. 28 shows representative data of a chemically modified siNA
construct (Stab 4/5, Table IV) targeting HBV site 1580 RNA compared to an
unstabilized siRNA construct in a dose response time course HBsAg assay.
The constructs were compared at different concentrations (5 nM, 10 nM, 25
nM, 50 nM, and 100 nM) over the course of nine days. Activity based on
HBsAg levels was determined at day 3, day 6, and day 9.

[0271] FIG. 29 shows representative data of a chemically modified siNA
construct (Stab 7/8, Table IV) targeting HBV site 1580 RNA compared to an
unstabilized siRNA construct in a dose response time course HBsAg assay.
The constructs were compared at different concentrations (5 nM, 10 nM, 25
nM, 50 nM, and 100 nM) over the course of nine days. SiNA activity based
on HBsAg levels was determined at day 3, day 6, and day 9.

[0272] FIG. 30 shows representative data of a chemically modified siNA
construct (Stab 7/11, Table IV) targeting HBV site 1580 RNA compared to
an unstabilized siRNA construct in a dose response time course HBsAg
assay. The constructs were compared at different concentrations (5 nM, 10
nM, 25 nM, 50 nM, and 100 nM) over the course of nine days. SiNA activity
based on HBsAg levels was determined at day 3, day 6, and day 9.

[0273] FIG. 31 shows representative data of a chemically modified siNA
construct (Stab 9/10, Table IV) targeting HBV site 1580 RNA compared to
an unstabilized siRNA construct in a dose response time course HBsAg
assay. The constructs were compared at different concentrations (5 nM, 10
nM, 25 nM, 50 nM, and 100 nM) over the course of nine days. SiNA activity
based on HBsAg levels was determined at day 3, day 6, and day 9.

[0275] FIG. 33 shows a non-limiting example of a dose response study
demonstrating the inhibition of viral replication of a HCV/poliovirus
chimera by siNA construct (29579/29586) at various concentrations (1 nM,
5 nM, 10 nM, and 25 nM) compared to control (29593/29600).

[0276] FIG. 34 shows a non-limiting example demonstrating the inhibition
of viral replication of a HCV/poliovirus chimera by a chemically modified
siRNA construct (30051/30053) compared to control construct
(30052/30054).

[0277] FIG. 35 shows a non-limiting example demonstrating the inhibition
of viral replication of a HCV/poliovirus chimera by a chemically modified
siRNA construct (30055/30057) compared to control construct
(30056/30058).

[0278] FIG. 36 shows a non-limiting example of several chemically modified
siRNA constructs targeting viral replication of an HCV/poliovirus chimera
at 10 nM treatment in comparison to a lipid control and an inverse siNA
control construct 29593/29600.

[0279] FIG. 37 shows a non-limiting example of several chemically modified
siRNA constructs targeting viral replication of a HCV/poliovirus chimera
at 25 nM treatment in comparison to a lipid control and an inverse siNA
control construct 29593/29600.

[0288] FIG. 46 shows a non-limiting example of a synthetic scheme for the
synthesis of a N-acetyl-D-galactosamine-2'-aminouridine phosphoramidite
conjugate of the invention.

[0289] FIG. 47 shows a non-limiting example of a synthetic scheme for the
synthesis of a N-acetyl-D-galactosamine-D-threoninol phosphoramidite
conjugate of the invention.

[0290] FIG. 48 shows a non-limiting example of a N-acetyl-D-galactosamine
siNA nucleic acid conjugate of the invention. W shown in the example
refers to a biodegradable linker, for example a nucleic acid dimer,
trimer, or tetramer comprising ribonucleotides and/or
deoxyribonucleotides. The siNA can be conjugated at the 3',5' or both 3'
and 5' ends of the sense strand of a double stranded siNA and/or the
3'-end of the antisense strand of the siNA. A single stranded siNA
molecule can be conjugated at the 3'-end of the siNA.

[0291] FIG. 49 shows a non-limiting example of a synthetic scheme for the
synthesis of a dodecanoic acid derived conjugate linker of the invention.

[0292] FIG. 50 shows a non-limiting example of a synthetic scheme for the
synthesis of an oxime linked nucleic acid/peptide conjugate of the
invention.

[0293] FIG. 51 shows non-limiting examples of phospholipid derived siNA
conjugates of the invention. CL shown in the examples refers to a
biodegradable linker, for example a nucleic acid dimer, trimer, or
tetramer comprising ribonucleotides and/or deoxyribonucleotides. The siNA
can be conjugated at the 3',5' or both 3' and 5' ends of the sense strand
of a double stranded siNA and/or the 3'-end of the antisense strand of
the siNA. A single stranded siNA molecule can be conjugated at the 3'-end
of the siNA.

[0294] FIG. 52 shows a non-limiting example of a synthetic scheme for
preparing a phospholipid derived siNA conjugates of the invention.

[0295] FIG. 53 shows a non-limiting example of a synthetic scheme for
preparing a poly-N-acetyl-D-galactosamine nucleic acid conjugate of the
invention.

[0296] FIG. 54 shows a non-limiting example of the synthesis of siNA
cholesterol conjugates of the invention using a phosphoramidite approach.

[0297] FIG. 55 shows a non-limiting example of the synthesis of siNA PEG
conjugates of the invention using NHS ester coupling.

[0298] FIG. 56 shows a non-limiting example of the synthesis of siNA
cholesterol conjugates of the invention using NHS ester coupling.

[0299] FIG. 57 shows a non-limiting example of various siNA cholesterol
conjugates of the invention.

[0300] FIG. 58 shows a non-limiting example of various siNA cholesterol
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a double
stranded siNA molecule.

[0301] FIG. 59 shows a non-limiting example of various siNA cholesterol
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a double
stranded siNA molecule.

[0302] FIG. 60 shows a non-limiting example of various siNA cholesterol
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a single
stranded siNA molecule.

[0303] FIG. 61 shows a non-limiting example of various siNA phospholipid
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a double
stranded siNA molecule.

[0304] FIG. 62 shows a non-limiting example of various siNA phospholipid
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a single
stranded siNA molecule.

[0305] FIG. 63 shows a non-limiting example of various siNA galactosamine
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a double
stranded siNA molecule.

[0306] FIG. 64 shows a non-limiting example of various siNA galactosamine
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a single
stranded siNA molecule.

[0307] FIG. 65 shows a non-limiting example of various generalized siNA
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a double
stranded siNA molecule. CONJ in the figure refers to any biologically
active compound or any other conjugate compound as described herein and
in the Formulae herein.

[0308] FIG. 66 shows a non-limiting example of various generalized siNA
conjugates of the invention in which various linker chemistries and/or
cleavable linkers can be utilized at different positions of a single
stranded siNA molecule. CONJ in the figure refers to any biologically
active compound or any other conjugate compound as described herein and
in the Formulae herein.

[0309] FIG. 67 shows a non-limiting example of the pharmacokinetic
distribution of intact siNA in liver after administration of conjugated
or unconjugated siNA molecules in mice.

[0310] FIG. 68 shows a non-limiting example of the activity of conjugated
siNA constructs compared to matched chemistry unconjugated siNA
constructs in an HBV cell culture system without the use of transfection
lipid. As shown in the Figure, siNA conjugates provide efficacy in cell
culture without the need for transfection reagent.

[0311] FIG. 69 shows a non-limiting example of a scheme for the synthesis
of a mono-galactosamine phosphoramidite of the invention that can be used
to generate galactosamine conjugated nucleic acid molecules.

[0312] FIG. 70 shows a non-limiting example of a scheme for the synthesis
of a tri-galactosamine phosphoramidite of the invention that can be used
to generate tri-galactosamine conjugated nucleic acid molecules.

[0313] FIG. 71 shows a non-limiting example of a scheme for the synthesis
of another tri-galactosamine phosphoramidite of the invention that can be
used to generate tri-galactosamine conjugated nucleic acid molecules.

[0314] FIG. 72 shows a non-limiting example of an alternate scheme for the
synthesis of a tri-galactosamine phosphoramidite of the invention that
can be used to generate tri-galactosamine conjugated nucleic acid
molecules.

[0315] FIG. 73 shows a non-limiting example of a scheme for the synthesis
of a cholesterol NHS ester of the invention that can be used to generate
cholesterol conjugated nucleic acid molecules.

[0318] FIG. 76 shows a non-limiting example of inhibition of VEGF induced
neovascularization in the rat corneal model. VEGFr1 site 349 active siNA
having "Stab 9/10" chemistry (Sirna #31270/31273) was tested for
inhibition of VEGF-induced angiogenesis at three different concentrations
(2.0 ug, 1.0 ug, and 0.1 μg dose response) as compared to a matched
chemistry inverted control siNA construct (Sirna #31276/31279) at each
concentration and a VEGF control in which no siNA was administered. As
shown in the figure, the active siNA construct having "Stab 9/10"
chemistry (Sirna #31270/31273) is highly effective in inhibiting
VEGF-induced angiogenesis in the rat corneal model compared to the
matched chemistry inverted control siNA at concentrations from 0.1 μg
to 2.0 ug.

[0328] FIG. 86 shows a non-limiting example of an assay screen of Stab 7/8
siNA constructs targeting various sites of HCV RNA in a replicon system
compared to untreated, lipid, and an inverted control. As shown in the
figure, several Stab 7/8 constructs were identified with potent anti-HCV
activity as shown by reduction in HCV RNA levels.

[0329] FIG. 87 shows a non-limiting example of an assay screen of Stab 7/8
siNA constructs targeting various sites of HBV RNA in HEpG2 cells
compared to untreated cells and an inverted control. As shown in the
figure, several Stab 7/8 constructs were identified with potent anti-HBV
activity as shown by reduction in HBV S antigen levels.

[0333] The discussion that follows discusses the proposed mechanism of RNA
interference mediated by short interfering RNA as is presently known, and
is not meant to be limiting and is not an admission of prior art.
Applicant demonstrates herein that chemically-modified short interfering
nucleic acids possess similar or improved capacity to mediate RNAi as do
siRNA molecules and are expected to possess improved stability and
activity in vivo; therefore, this discussion is not meant to be limited
to siRNA only and can be applied to siNA as a whole. By "improved
capacity to mediate RNAi" or "improved RNAi activity" is meant to include
RNAi activity measured in vitro and/or in vivo where the RNAi activity is
a reflection of both the ability of the siNA to mediate RNAi and the
stability of the siNAs of the invention. In this invention, the product
of these activities can be increased in vitro and/or in vivo compared to
an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In
some cases, the activity or stability of the siNA molecule can be
decreased (i.e., less than ten-fold), but the overall activity of the
siNA molecule is enhanced in vitro and/or in vivo.

[0334] RNA interference refers to the process of sequence specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et
al., 1998, Nature, 391, 806). The corresponding process in plants is
commonly referred to as post-transcriptional gene silencing or RNA
silencing and is also referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent the
expression of foreign genes which is commonly shared by diverse flora and
phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from
foreign gene expression may have evolved in response to the production of
double-stranded RNAs (dsRNAs) derived from viral infection or the random
integration of transposon elements into a host genome via a cellular
response that specifically destroys homologous single-stranded RNA or
viral genomic RNA. The presence of dsRNA in cells triggers the RNAi
response though a mechanism that has yet to be fully characterized. This
mechanism appears to be different from the interferon response that
results from dsRNA-mediated activation of protein kinase PKR and
2',5'-oligoadenylate synthetase resulting in non-specific cleavage of
mRNA by ribonuclease L.

[0335] The presence of long dsRNAs in cells stimulates the activity of a
ribonuclease III enzyme referred to as Dicer. Dicer is involved in the
processing of the dsRNA into short pieces of dsRNA known as short
interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).
Short interfering RNAs derived from Dicer activity are typically about 21
to about 23 nucleotides in length and comprise about 19 base pair
duplexes. Dicer has also been implicated in the excision of 21- and
22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of
conserved structure that are implicated in translational control
(Hutvagner et al., 2001, Science, 293, 834). The RNAi response also
features an endonuclease complex containing a siRNA, commonly referred to
as an RNA-induced silencing complex (RISC), which mediates cleavage of
single-stranded RNA having sequence homologous to the siRNA. Cleavage of
the target RNA takes place in the middle of the region complementary to
the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes
Dev., 15, 188). In addition, RNA interference can also involve small RNA
(e.g., micro-RNA or miRNA) mediated gene silencing, presumably though
cellular mechanisms that regulate chromatin structure and thereby prevent
transcription of target gene sequences (see for example Allshire, 2002,
Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837;
Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science,
297, 2232-2237). As such, siNA molecules of the invention can be used to
mediate gene silencing via interaction with RNA transcripts or
alternately by interaction with particular gene sequences, wherein such
interaction results in gene silencing either at the transcriptional level
or post-transcriptional level.

[0336] RNAi has been studied in a variety of systems. Fire et al., 1998,
Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny
and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by
dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe
RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,
Nature, 411, 494, describe RNAi induced by introduction of duplexes of
synthetic 21-nucleotide RNAs in cultured mammalian cells including human
embryonic kidney and HeLa cells. Recent work in Drosophila embryonic
lysates has revealed certain requirements for siRNA length, structure,
chemical composition, and sequence that are essential to mediate
efficient RNAi activity. These studies have shown that 21 nucleotide
siRNA duplexes are most active when containing two 2-nucleotide
3'-terminal nucleotide overhangs. Furthermore, substitution of one or
both siRNA strands with 2'-deoxy or 2'-O-methyl nucleotides abolishes
RNAi activity, whereas substitution of 3'-terminal siRNA nucleotides with
deoxy nucleotides was shown to be tolerated. Mismatch sequences in the
center of the siRNA duplex were also shown to abolish RNAi activity. In
addition, these studies also indicate that the position of the cleavage
site in the target RNA is defined by the 5'-end of the siRNA guide
sequence rather than the 3'-end (Elbashir et al., 2001, EMBO J., 20,
6877). Other studies have indicated that a 5'-phosphate on the
target-complementary strand of a siRNA duplex is required for siRNA
activity and that ATP is utilized to maintain the 5'-phosphate moiety on
the siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNA
molecules lacking a 5'-phosphate are active when introduced exogenously,
suggesting that 5'-phosphorylation of siRNA constructs may occur in vivo.

Synthesis of Nucleic Acid Molecules

[0337] Synthesis of nucleic acids greater than 100 nucleotides in length
is difficult using automated methods, and the therapeutic cost of such
molecules is prohibitive. In this invention, small nucleic acid motifs
("small" refers to nucleic acid motifs no more than 100 nucleotides in
length, preferably no more than 80 nucleotides in length, and most
preferably no more than 50 nucleotides in length; e.g., individual siNA
oligonucleotide sequences or siNA sequences synthesized in tandem) are
preferably used for exogenous delivery. The simple structure of these
molecules increases the ability of the nucleic acid to invade targeted
regions of protein and/or RNA structure. Exemplary molecules of the
instant invention are chemically synthesized, and others can similarly be
synthesized.

[0338] Oligonucleotides (e.g., certain modified oligonucleotides or
portions of oligonucleotides lacking ribonucleotides) are synthesized
using protocols known in the art, for example as described in Caruthers
et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,
International PCT Publication No. WO 99/54459, Wincott et al., 1995,
Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.
Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and
Brennan, U.S. Pat. No. 6,001,311. All of these references are
incorporated herein by reference. The synthesis of oligonucleotides makes
use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a
non-limiting example, small scale syntheses are conducted on a 394
Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol
with a 2.5 min coupling step for 2'-β-methylated nucleotides and a
45 second coupling step for 2'-deoxy nucleotides or 2'-deoxy-2'-fluoro
nucleotides. Table V outlines the amounts and the contact times of the
reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2
μmol scale can be performed on a 96-well plate synthesizer, such as
the instrument produced by Protogene (Palo Alto, Calif.) with minimal
modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6
μmol) of 2'-O-methyl phosphoramidite and a 105-fold excess of S-ethyl
tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling
cycle of 2'-O-methyl residues relative to polymer-bound 5'-hydroxyl. A
22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite
and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol)
can be used in each coupling cycle of deoxy residues relative to
polymer-bound 5'-hydroxyl. Average coupling yields on the 394 Applied
Biosystems, Inc. synthesizer, determined by colorimetric quantitation of
the trityl fractions, are typically 97.5-99%. Other oligonucleotide
synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer
include the following: detritylation solution is 3% TCA in methylene
chloride (ABI); capping is performed with 16% N-methyl imidazole in THF
(ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and
oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF
(PERSEPTIVE®). Burdick & Jackson Synthesis Grade acetonitrile is used
directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in
acetonitrile) is made up from the solid obtained from American
International Chemical, Inc. Alternately, for the introduction of
phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one
1,1-dioxide, 0.05 M in acetonitrile) is used.

[0339] Deprotection of the DNA-based oligonucleotides is performed as
follows: the polymer-bound trityl-on oligoribonucleotide is transferred
to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous
methylamine (1 mL) at 65° C. for 10 minutes. After cooling to
-20° C., the supernatant is removed from the polymer support. The
support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1,
vortexed and the supernatant is then added to the first supernatant. The
combined supernatants, containing the oligoribonucleotide, are dried to a
white powder.

[0340] The method of synthesis used for RNA including certain siNA
molecules of the invention follows the procedure as described in Usman et
al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic
Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23,
2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use
of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a
non-limiting example, small scale syntheses are conducted on a 394
Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol
with a 7.5 min coupling step for alkylsilyl protected nucleotides and a
2.5 min coupling step for 2'-O-methylated nucleotides. Table V outlines
the amounts and the contact times of the reagents used in the synthesis
cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a
96-well plate synthesizer, such as the instrument produced by Protogene
(Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold
excess (60 μL of 0.11 M=6.6 μmol) of 2'-O-methyl phosphoramidite
and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol)
can be used in each coupling cycle of 2'-O-methyl residues relative to
polymer-bound 5'-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2
μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold
excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used
in each coupling cycle of ribo residues relative to polymer-bound
5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.
synthesizer, determined by colorimetric quantitation of the trityl
fractions, are typically 97.5-99%. Other oligonucleotide synthesis
reagents for the 394 Applied Biosystems, Inc. synthesizer include the
following: detritylation solution is 3% TCA in methylene chloride (ABI);
capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%
acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is
16.9 mM I2, 49 mM pyridine, 9% water in THF (PERSEPTIVE®).
Burdick & Jackson Synthesis Grade acetonitrile is used directly from the
reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is
made up from the solid obtained from American International Chemical,
Inc. Alternately, for the introduction of phosphorothioate linkages,
Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in
acetonitrile) is used.

[0341] Deprotection of the RNA is performed using either a two-pot or
one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on
oligoribonucleotide is transferred to a 4 mL glass screw top vial and
suspended in a solution of 40% aq. methylamine (1 mL) at 65° C.
for 10 minutes. After cooling to -20° C., the supernatant is
removed from the polymer support. The support is washed three times with
1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added
to the first supernatant. The combined supernatants, containing the
oligoribonucleotide, are dried to a white powder. The base deprotected
oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300
μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1
mL TEA3HF to provide a 1.4 M HF concentration) and heated to 65°
C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.

[0342] Alternatively, for the one-pot protocol, the polymer-bound
trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top
vial and suspended in a solution of 33% ethanolic methylamine/DMSO:1/1
(0.8 mL) at 65° C. for 15 minutes. The vial is brought to room
temperature TEA3HF (0.1 mL) is added and the vial is heated at 65°
C. for 15 minutes. The sample is cooled at -20° C. and then
quenched with 1.5 M NH4HCO3.

[0343] For purification of the trityl-on oligomers, the quenched
NH4HCO3 solution is loaded onto a C-18 containing cartridge
that had been prewashed with acetonitrile followed by 50 mM TEAA. After
washing the loaded cartridge with water, the RNA is detritylated with
0.5% TFA for 13 minutes. The cartridge is then washed again with water,
salt exchanged with 1 M NaCl and washed with water again. The
oligonucleotide is then eluted with 30% acetonitrile.

[0344] The average stepwise coupling yields are typically >98% (Wincott
et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill
in the art will recognize that the scale of synthesis can be adapted to
be larger or smaller than the example described above including but not
limited to 96-well format.

[0346] The siNA molecules of the invention can also be synthesized via a
tandem synthesis methodology as described in Example 1 herein, wherein
both siNA strands are synthesized as a single contiguous oligonucleotide
fragment or strand separated by a cleavable linker which is subsequently
cleaved to provide separate siNA fragments or strands that hybridize and
permit purification of the siNA duplex. The linker can be a
polynucleotide linker or a non-nucleotide linker. The tandem synthesis of
siNA as described herein can be readily adapted to both
multiwell/multiplate synthesis platforms such as 96 well or similarly
larger multi-well platforms. The tandem synthesis of siNA as described
herein can also be readily adapted to large scale synthesis platforms
employing batch reactors, synthesis columns and the like.

[0347] A siNA molecule can also be assembled from two distinct nucleic
acid strands or fragments wherein one fragment includes the sense region
and the second fragment includes the antisense region of the RNA
molecule.

[0348] The nucleic acid molecules of the present invention can be modified
extensively to enhance stability by modification with nuclease resistant
groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H
(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,
1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified
by gel electrophoresis using general methods or can be purified by high
pressure liquid chromatography (HPLC; see Wincott et al., supra, the
totality of which is hereby incorporated herein by reference) and
re-suspended in water.

[0349] In another aspect of the invention, siNA molecules of the invention
are expressed from transcription units inserted into DNA or RNA vectors.
The recombinant vectors can be DNA plasmids or viral vectors. siNA
expressing viral vectors can be constructed based on, but not limited to,
adeno-associated virus, retrovirus, adenovirus, or alphavirus. The
recombinant vectors capable of expressing the siNA molecules can be
delivered as described herein, and persist in target cells.
Alternatively, viral vectors can be used that provide for transient
expression of siNA molecules.

[0352] While chemical modification of oligonucleotide internucleotide
linkages with phosphorothioate, phosphorodithioate, and/or
5'-methylphosphonate linkages improves stability, excessive modifications
can cause some toxicity or decreased activity. Therefore, when designing
nucleic acid molecules, the amount of these internucleotide linkages
should be minimized. The reduction in the concentration of these linkages
should lower toxicity, resulting in increased efficacy and higher
specificity of these molecules.

[0353] Short interfering nucleic acid (siNA) molecules having chemical
modifications that maintain or enhance activity are provided. Such a
nucleic acid is also generally more resistant to nucleases than an
unmodified nucleic acid. Accordingly, the in vitro and/or in vivo
activity should not be significantly lowered. In cases in which
modulation is the goal, therapeutic nucleic acid molecules delivered
exogenously should optimally be stable within cells until translation of
the target RNA has been modulated long enough to reduce the levels of the
undesirable protein. This period of time varies between hours to days
depending upon the disease state. Improvements in the chemical synthesis
of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677;
Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by
reference herein)) have expanded the ability to modify nucleic acid
molecules by introducing nucleotide modifications to enhance their
nuclease stability, as described above.

[0354] In one embodiment, nucleic acid molecules of the invention include
one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp
nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein
the modifications confer the ability to hydrogen bond both Watson-Crick
and Hoogsteen faces of a complementary guanine within a duplex, see for
example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A
single G-clamp analog substitution within an oligonucleotide can result
in substantially enhanced helical thermal stability and mismatch
discrimination when hybridized to complementary oligonucleotides. The
inclusion of such nucleotides in nucleic acid molecules of the invention
results in both enhanced affinity and specificity to nucleic acid
targets, complementary sequences, or template strands. In another
embodiment, nucleic acid molecules of the invention include one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA "locked nucleic
acid" nucleotides such as a 2',4'-C methylene bicyclo nucleotide (see for
example Wengel et al., International PCT Publication No. WO 00/66604 and
WO 99/14226).

[0355] In another embodiment, the invention features conjugates and/or
complexes of siNA molecules of the invention. Such conjugates and/or
complexes can be used to facilitate delivery of siNA molecules into a
biological system, such as a cell. The conjugates and complexes provided
by the instant invention can impart therapeutic activity by transferring
therapeutic compounds across cellular membranes, altering the
pharmacokinetics, and/or modulating the localization of nucleic acid
molecules of the invention. The present invention encompasses the design
and synthesis of novel conjugates and complexes for the delivery of
molecules, including, but not limited to, small molecules, lipids,
cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,
antibodies, toxins, negatively charged polymers and other polymers, for
example, proteins, peptides, hormones, carbohydrates, polyethylene
glycols, or polyamines, across cellular membranes. In general, the
transporters described are designed to be used either individually or as
part of a multi-component system, with or without degradable linkers.
These compounds are expected to improve delivery and/or localization of
nucleic acid molecules of the invention into a number of cell types
originating from different tissues, in the presence or absence of serum
(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the
molecules described herein can be attached to biologically active
molecules via linkers that are biodegradable, such as biodegradable
nucleic acid linker molecules.

[0356] In one embodiment, the invention features a compound having Formula
1:

##STR00008##

[0357] wherein each R1, R3, R4, R5, R6, R7
and R8 is independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, or a protecting group, each "n" is independently an
integer from 0 to about 200, R12 is a straight or branched chain
alkyl, substituted alkyl, aryl, or substituted aryl, and R2 is a
siNA molecule or a portion thereof.

[0358] In one embodiment, the invention features a compound having Formula
2:

##STR00009##

[0359] wherein each R3, R4, R5, R6 and R7 is
independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
or a protecting group, each "n" is independently an integer from 0 to
about 200, R12 is a straight or branched chain alkyl, substituted
alkyl, aryl, or substituted aryl, and R2 is a siNA molecule or a
portion thereof.

[0360] In one embodiment, the invention features a compound having Formula
3:

##STR00010##

[0361] wherein each R1, R3, R4, R5, R6 and
R7 is independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, or a protecting group, each "n" is independently an
integer from 0 to about 200, R12 is a straight or branched chain
alkyl, substituted alkyl, aryl, or substituted aryl, and R2 is a
siNA molecule or a portion thereof.

[0362] In one embodiment, the invention features a compound having Formula
4:

##STR00011##

[0363] wherein each R3, R4, R5, R6 and R7 is
independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
or a protecting group, each "n" is independently an integer from 0 to
about 200, R2 is a siNA molecule or a portion thereof, and R13
is an amino acid side chain.

[0364] In one embodiment, the invention features a compound having Formula
5:

##STR00012##

[0365] wherein each R1 and R4 is independently a protecting
group or hydrogen, each R3, R5, R6, R7 and R8 is
independently hydrogen, alkyl or nitrogen protecting group, each "n" is
independently an integer from 0 to about 200, R12 is a straight or
branched chain alkyl, substituted alkyl, aryl, or substituted aryl, and
each R9 and R10 is independently a nitrogen containing group,
cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

[0366] In one embodiment, the invention features a compound having Formula
6:

##STR00013##

[0367] wherein each R4, R5, R6 and R7 is independently
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or a
protecting group, R2 is a siNA molecule or a portion thereof, each
"n" is independently an integer from 0 to about 200, and L is a
degradable linker.

[0368] In one embodiment, the invention features a compound having Formula
7:

##STR00014##

[0369] wherein each R1, R3, R4, R5, R6 and
R7 is independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, or a protecting group, each "n" is independently an
integer from 0 to about 200, R12 is a straight or branched chain
alkyl, substituted alkyl, aryl, or substituted aryl, and R2 is a
siNA molecule or a portion thereof.

[0370] In one embodiment, the invention features a compound having Formula
8:

##STR00015##

[0371] wherein each R1 and R4 is independently a protecting
group or hydrogen, each R3, R5, R6 and R7 is
independently hydrogen, alkyl or nitrogen protecting group, each "n" is
independently an integer from 0 to about 200, R12 is a straight or
branched chain alkyl, substituted alkyl, aryl, or substituted aryl, and
each R9 and R10 is independently a nitrogen containing group,
cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

[0372] In one embodiment, R13 of a compound of the invention
comprises an alkylamino or an alkoxy group, for example, --CH2O-- or
--CH(CH2)CH2O--.

[0373] In another embodiment, R12 of a compound of the invention is
an alkylhyrdroxyl, for example, --(CH2)nOH, where n comprises
an integer from about 1 to about 10.

[0374] In another embodiment, L of Formula 6 of the invention comprises
serine, threonine, or a photolabile linkage.

[0375] In one embodiment, R9 of a compound of the invention comprises
a phosphorus protecting group, for example --OCH2CH2CN
(oxyethylcyano).

[0376] In one embodiment, R10 of a compound of the invention
comprises a nitrogen containing group, for example, --N(R14) wherein
R14 is a straight or branched chain alkyl having from about 1 to
about 10 carbons.

[0377] In another embodiment, R10 of a compound of the invention
comprises a heterocycloalkyl or heterocycloalkenyl ring containing from
about 4 to about 7 atoms, and having from about 1 to about 3 heteroatoms
comprising oxygen, nitrogen, or sulfur.

[0378] In another embodiment, R1 of a compound of the invention
comprises an acid labile protecting group, such as a trityl or
substituted trityl group, for example, a dimethoxytrityl or
mono-methoxytrityl group.

[0379] In another embodiment, R4 of a compound of the invention
comprises a tert-butyl, Fm (fluorenyl-methoxy), or allyl group.

[0380] In one embodiment, R6 of a compound of the invention comprises
a TFA (trifluoracetyl) group.

[0381] In another embodiment, R3, R5, R7 and R8 of a
compound of the invention are independently hydrogen.

[0382] In one embodiment, R7 of a compound of the invention is
independently isobutyryl, dimethylformamide, or hydrogen.

[0383] In another embodiment, R12 of a compound of the invention
comprises a methyl group or ethyl group.

[0384] In one embodiment, the invention features a compound having Formula
27:

##STR00016##

[0385] wherein "n" is an integer from about 0 to about 20, R4 is H or
a cationic salt, X is a siNA molecule or a portion thereof, and R24
is a sulfur containing leaving group, for example a group comprising:

##STR00017##

[0386] In one embodiment, the invention features a compound having Formula
39:

##STR00018##

[0387] wherein "n" is an integer from about 0 to about 20, X is a siNA
molecule or a portion thereof, and P is a phosphorus containing group.

[0388] In another embodiment, a thiol containing linker of the invention
is a compound having Formula 41:

##STR00019##

[0389] wherein "n" is an integer from about 0 to about 20, P is a
phosphorus containing group, for example a phosphine, phosphite, or
phosphate, and R24 is any alkyl, substituted alkyl, alkoxy, aryl,
substituted aryl, alkenyl, substituted alkenyl, alkynyl, or substituted
alkynyl group with or without additional protecting groups.

[0390] In one embodiment, the invention features a compound having Formula
43:

##STR00020##

[0391] wherein X comprises a siNA molecule or portion thereof; W comprises
a degradable nucleic acid linker; Y comprises a linker molecule or amino
acid that can be present or absent; Z comprises H, OH, O-alkyl, SH,
S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino,
substituted amino, nucleotide, nucleoside, nucleic acid, oligonucleotide,
amino acid, peptide, protein, lipid, phospholipid, or label; n is an
integer from about 1 to about 100; and N' is an integer from about 1 to
about 20. In another embodiment, W is selected from the group consisting
of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate
ester linkage.

[0392] In another embodiment, the invention features a compound having
Formula 44:

##STR00021##

[0393] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent; n is
an integer from about 1 to about 50, and PEG represents a compound having
Formula 45:

[0395] In another embodiment, the invention features a compound having
Formula 46:

##STR00023##

[0396] wherein X comprises a siNA molecule or portion thereof; each W
independently comprises linker molecule or chemical linkage that can be
present or absent, Y comprises a linker molecule or chemical linkage that
can be present or absent; and PEG represents a compound having Formula
45:

[0398] In one embodiment, the invention features a compound having Formula
47:

##STR00025##

[0399] wherein X comprises a siNA molecule or portion thereof; each W
independently comprises a linker molecule or chemical linkage that can be
the same or different and can be present or absent, Y comprises a linker
molecule that can be present or absent; each Q independently comprises a
hydrophobic group or phospholipid; each R1, R2, R3, and R4 independently
comprises O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, and n is an integer from about 1 to
about 10. In another embodiment, W is selected from the group consisting
of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate
ester linkage.

[0400] In another embodiment, the invention features a compound having
Formula 48:

##STR00026##

[0401] wherein X comprises a siNA molecule or portion thereof; each W
independently comprises a linker molecule or chemical linkage that can be
present or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or
substituted N, and B represents a lipophilic group, for example a
saturated or unsaturated linear, branched, or cyclic alkyl group,
cholesterol, or a derivative thereof. In another embodiment, W is
selected from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.

[0402] In another embodiment, the invention features a compound having
Formula 49:

##STR00027##

[0403] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, Y
comprises a linker molecule that can be present or absent; each R1, R2,
R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and B
represents a lipophilic group, for example a saturated or unsaturated
linear, branched, or cyclic alkyl group, cholesterol, or a derivative
thereof. In another embodiment, W is selected from the group consisting
of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate
ester linkage.

[0404] In another embodiment, the invention features a compound having
Formula 50:

##STR00028##

[0405] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, Y
comprises a linker molecule or chemical linkage that can be present or
absent; and each Q independently comprises a hydrophobic group or
phospholipid. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.

[0406] In one embodiment, the invention features a compound having Formula
51:

##STR00029##

[0407] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent; Y
comprises a linker molecule or amino acid that can be present or absent;
Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl,
substituted aryl, amino, substituted amino, nucleotide, nucleoside,
nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,
phospholipid, or label; SG comprises a sugar, for example galactose,
galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or
fucose and the respective D or L, alpha or beta isomers, and n is an
integer from about 1 to about 20. In another embodiment, W is selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.

[0408] In another embodiment, the invention features a compound having
Formula 52:

[0410] In another embodiment, the invention features a compound having
Formula 53:

##STR00031##

[0411] wherein B comprises H, a nucleoside base, or a non-nucleosidic base
with or without protecting groups; each R1 independently comprises O, N,
S, alkyl, or substituted N; each R2 independently comprises O, OH, H,
alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or a
phosphorus containing group; each R3 independently comprises N or O--N,
each R4 independently comprises O, CH2, S, sulfone, or sulfoxy; X
comprises H, a removable protecting group, a siNA molecule or a portion
thereof; W comprises a linker molecule or chemical linkage that can be
present or absent; SG comprises a sugar, for example galactose,
galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, or
fucose and the respective D or L, alpha or beta isomers, each n is
independently an integer from about 1 to about 50; and N' is an integer
from about 1 to about 10. In another embodiment, W is selected from the
group consisting of amide, phosphate, phosphate ester, phosphoramidate,
or thiophosphate ester linkage.

[0412] In another embodiment, the invention features a compound having
Formula 54:

##STR00032##

[0413] wherein B comprises H, a nucleoside base, or a non-nucleosidic base
with or without protecting groups; each R1 independently comprises O, OH,
H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or a
phosphorus containing group; X comprises H, a removable protecting group,
a siNA molecule or a portion thereof; W comprises a linker molecule or
chemical linkage that can be present or absent; and SG comprises a sugar,
for example galactose, galactosamine, N-acetyl-galactosamine, glucose,
mannose, fructose, or fucose and the respective D or L, alpha or beta
isomers. In another embodiment, W is selected from the group consisting
of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate
ester linkage.

[0414] In one embodiment, the invention features a compound having Formula
55:

##STR00033##

[0415] wherein each R1 independently comprises O, N, S, alkyl, or
substituted N; each R2 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or a phosphorus
containing group; each R3 independently comprises H, OH, alkyl,
substituted alkyl, or halo; X comprises H, a removable protecting group,
a siNA molecule or a portion thereof; W comprises a linker molecule or
chemical linkage that can be present or absent; SG comprises a sugar, for
example galactose, galactosamine, N-acetyl-galactosamine, glucose,
mannose, fructose, or fucose and the respective D or L, alpha or beta
isomers, each n is independently an integer from about 1 to about 50; and
N' is an integer from about 1 to about 100. In another embodiment, W is
selected from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.

[0416] In another embodiment, the invention features a compound having
Formula 56:

##STR00034##

[0417] wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or a
phosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,
halo, S, N, substituted N, or a phosphorus containing group; X comprises
H, a removable protecting group, a siNA molecule or a portion thereof; W
comprises a linker molecule or chemical linkage that can be present or
absent; SG comprises a sugar, for example galactose, galactosamine,
N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and the
respective D or L, alpha or beta isomers, and each n is independently an
integer from about 0 to about 20. In another embodiment, W is selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.

[0418] In another embodiment, the invention features a compound having
Formula 57:

##STR00035## [0419] wherein R1 can include the groups:

[0419] ##STR00036## [0420] and wherein R2 can include the groups:

##STR00037##

[0421] and wherein Tr is a removable protecting group, for example a
trityl, monomethoxytrityl, or dimethoxytrityl; SG comprises a sugar, for
example galactose, galactosamine, N-acetyl-galactosamine, glucose,
mannose, fructose, or fucose and the respective D or L, alpha or beta
isomers, and n is an integer from about 1 to about 20.

[0422] In one embodiment, compounds having Formula 52, 53, 54, 55, 56, and
57 are featured wherein each nitrogen adjacent to a carbonyl can
independently be substituted for a carbonyl adjacent to a nitrogen or
each carbonyl adjacent to a nitrogen can be substituted for a nitrogen
adjacent to a carbonyl.

[0423] In another embodiment, the invention features a compound having
Formula 58:

##STR00038##

[0424] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent; Y
comprises a linker molecule or amino acid that can be present or absent;
V comprises a signal protein or peptide, for example Human serum albumin
protein, Antennapedia peptide, Kaposi fibroblast growth factor peptide,
Caiman crocodylus Ig(5) light chain peptide, HIV envelope glycoprotein
gp41 peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelope
glycoprotein peptide, or transportan A peptide; each n is independently
an integer from about 1 to about 50; and N' is an integer from about 1 to
about 100. In another embodiment, W is selected from the group consisting
of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate
ester linkage.

[0425] In another embodiment, the invention features a compound having
Formula 59:

[0427] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted
alkyl, aryl, substituted aryl, amino, substituted amino, a removable
protecting group, a siNA molecule or a portion thereof; and n is an
integer from about 1 to about 100. In another embodiment, W is selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.

[0428] In another embodiment, the invention features a compound having
Formula 60:

##STR00041## [0429] wherein R1 can include the groups:

[0429] ##STR00042## [0430] and wherein R2 can include the groups:

##STR00043##

[0431] and wherein Tr is a removable protecting group, for example a
trityl, monomethoxytrityl, or dimethoxytrityl; n is an integer from about
1 to about 50; and R8 is a nitrogen protecting group, for example a
phthaloyl, trifluoroacetyl, FMOC, or monomethoxytrityl group.

[0432] In another embodiment, the invention features a compound having
Formula 61:

##STR00044##

[0433] wherein X comprises a siNA molecule or portion thereof; each W
independently comprises a linker molecule or chemical linkage that can be
the same or different and can be present or absent, Y comprises a linker
molecule that can be present or absent; each 5 independently comprises a
signal protein or peptide, for example Human serum albumin protein,
Antennapedia peptide, Kaposi fibroblast growth factor peptide, Caiman
crocodylus Ig(5) light chain peptide, HIV envelope glycoprotein gp41
peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelope glycoprotein
peptide, or transportan A peptide; each R1, R2, R3, and R4 independently
comprises O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,
S-alkylcyano, N or substituted N, and n is an integer from about 1 to
about 10. In another embodiment, W is selected from the group consisting
of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate
ester linkage.

[0434] In another embodiment, the invention features a compound having
Formula 62:

[0438] In another embodiment, the invention features a compound having
Formula 64:

##STR00047##

[0439] wherein X comprises a siNA molecule or portion thereof; each W
independently comprises a linker molecule or chemical linkage that can be
present or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or
substituted N, A comprises a nitrogen containing group, and B comprises a
lipophilic group. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.

[0440] In another embodiment, the invention features a compound having
Formula 65:

##STR00048##

[0441] wherein X comprises a siNA molecule or portion thereof; each W
independently comprises a linker molecule or chemical linkage that can be
present or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or
substituted N, RV comprises the lipid or phospholipid component of any of
Formulae 47-50, and R6 comprises a nitrogen containing group. In another
embodiment, W is selected from the group consisting of amide, phosphate,
phosphate ester, phosphoramidate, or thiophosphate ester linkage.

[0442] In another embodiment, the invention features a compound having
Formula 92:

[0450] In another embodiment, the invention features a compound having
Formula 99:

##STR00053##

[0451] wherein X comprises a siNA molecule or portion thereof; each W
independently comprises a linker molecule or chemical linkage that can be
present or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or
substituted N, and SG comprises a sugar, for example galactose,
galactosamine, N-acetyl-galactosamine or branched derivative thereof,
glucose, mannose, fructose, or fucose and the respective D or L, alpha or
beta isomers. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.

[0452] In another embodiment, the invention features a compound having
Formula 100:

##STR00054##

[0453] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, Y
comprises a linker molecule that can be present or absent; each R1, R2,
R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and SG
comprises a sugar, for example galactose, galactosamine,
N-acetyl-galactosamine or branched derivative thereof, glucose, mannose,
fructose, or fucose and the respective D or L, alpha or beta isomers. In
another embodiment, W is selected from the group consisting of amide,
phosphate, phosphate ester, phosphoramidate, or thiophosphate ester
linkage.

[0454] In one embodiment, the SG component of any compound having Formulae
99 or 100 comprises a compound having Formula 101:

##STR00055##

[0455] wherein Y comprises a linker molecule or chemical linkage that can
be present or absent and each R7 independently comprises an acyl group
that can be present or absent, for example a acetyl group.

[0456] In one embodiment, the W-SG component of a compound having Formulae
99 comprises a compound having Formula 102:

##STR00056##

[0457] wherein R2 comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylhalo, S, N, substituted N, a protecting group, or another compound
having Formula 102; R1 independently H, OH, alkyl, substituted alkyl, or
halo and each R7 independently comprises an acyl group that can be
present or absent, for example a acetyl group, and R3 comprises 0 or R3
in Formula 99, and n is an integer from about 1 to about 20.

[0458] In one embodiment, the W-SG component of a compound having Formulae
99 comprises a compound having Formula 103:

##STR00057##

[0459] wherein R1 comprises H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S,
N, substituted N, a protecting group, or another compound having Formula
103; each R7 independently comprises an acyl group that can be present or
absent, for example a acetyl group, and R3 comprises H or R3 in Formula
99, and each n is independently an integer from about 1 to about 20.

[0460] In one embodiment, the invention features a compound having Formula
104:

##STR00058##

[0461] wherein R3 comprises H, OH, amino, substituted amino, nucleotide,
nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,
lipid, phospholipid, label, or a portion thereof, or OR5 where R5 a
removable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,
O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7
independently comprises an acyl group that can be present or absent, for
example a acetyl group, and each n is independently an integer from about
1 to about 20, and wherein R1 can include the groups:

##STR00059##

[0462] and wherein R2 can include the groups:

##STR00060##

[0463] In one embodiment, the invention features a compound having Formula
105:

##STR00061##

[0464] wherein X comprises a siNA molecule or a portion thereof, R2
comprises O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N,
substituted N, a protecting group, or a nucleotide, polynucleotide, or
oligonucleotide or a portion thereof; R1 independently H, OH, alkyl,
substituted alkyl, or halo and each R7 independently comprises an acyl
group that can be present or absent, for example a acetyl group, and n is
an integer from about 1 to about 20.

[0465] In one embodiment, the invention features a compound having Formula
106:

##STR00062##

[0466] wherein X comprises a siNA molecule or a portion thereof, R1
comprises H, OH, amino, substituted amino, nucleotide, nucleoside,
nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,
phospholipid, label, or a portion thereof, or OR5 where R5 a removable
protecting group, each R7 independently comprises an acyl group that can
be present or absent, for example a acetyl group, and each n is
independently an integer from about 1 to about 20

[0467] In another embodiment, the invention features a compound having
Formula 107:

##STR00063##

[0468] wherein X comprises a siNA molecule or portion thereof; each W
independently comprises a linker molecule or chemical linkage that can be
present or absent, Y comprises a linker molecule that can be present or
absent; each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,
alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or
substituted N, and Cholesterol comprises cholesterol or an analog,
derivative, or metabolite thereof. In another embodiment, W is selected
from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.

[0469] In another embodiment, the invention features a compound having
Formula 108:

##STR00064##

[0470] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, Y
comprises a linker molecule that can be present or absent; each R1, R2,
R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,
O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and
Cholesterol comprises cholesterol or an analog, derivative, or metabolite
thereof. In another embodiment, W is selected from the group consisting
of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate
ester linkage.

[0471] In one embodiment, the W-Cholesterol component of a compound having
Formula 107 comprises a compound having Formula 109:

##STR00065##

[0472] wherein R3 comprises R3 as described in Formula 107, and n is
independently an integer from about 1 to about 20.

[0473] In one embodiment, the invention features a compound having Formula
110:

##STR00066##

[0474] wherein R4 comprises O, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S,
S-alkyl, S-alkylcyano, N or substituted N, each n is independently an
integer from about 1 to about 20, and

[0475] wherein R1 can include the groups:

##STR00067##

[0476] and wherein R2 can include the groups:

##STR00068##

[0477] In one embodiment, the invention features a compound having Formula
III:

##STR00069##

[0478] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, and
n is an integer from about 1 to about 20. In another embodiment, W is
selected from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.

[0479] In one embodiment, the invention features a compound having Formula
112:

##STR00070##

[0480] wherein n is an integer from about 1 to about 20. In another
embodiment, a compound having Formula 112 is used to generate a compound
having Formula III via NHS ester mediated coupling with a biologically
active molecule, such as a siNA molecule or a portion thereof. In a
non-limiting example, the NHS ester coupling can be effectuated via
attachment to a free amine present in the siNA molecule, such as an amino
linker molecule present on a nucleic acid sugar (e.g. 2'-amino linker) or
base (e.g., C5 alkyl amine linker) component of the siNA molecule.

[0481] In one embodiment, the invention features a compound having Formula
113:

[0484] In another embodiment, a compound having Formula 113 is used to
generate a compound having Formula III via phosphoramidite mediated
coupling with a biologically active molecule, such as a siNA molecule or
a portion thereof.

[0485] In one embodiment, the invention features a compound having Formula
114:

##STR00074##

[0486] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, and
n is an integer from about 1 to about 20. In another embodiment, W is
selected from the group consisting of amide, phosphate, phosphate ester,
phosphoramidate, or thiophosphate ester linkage.

[0487] In one embodiment, the invention features a compound having Formula
115:

##STR00075##

[0488] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, R3
comprises H, OH, amino, substituted amino, nucleotide, nucleoside,
nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,
phospholipid, label, or a portion thereof, or OR5 where R5 a removable
protecting group, and each n is independently an integer from about 1 to
about 20. In another embodiment, W is selected from the group consisting
of amide, phosphate, phosphate ester, phosphoramidate, or thiophosphate
ester linkage.

[0489] In one embodiment, the invention features a compound having Formula
116:

[0492] In another embodiment, a compound having Formula 116 is used to
generate a compound having Formula 114 or 115 via phosphoramidite
mediated coupling with a biologically active molecule, such as a siNA
molecule or a portion thereof.

[0493] In one embodiment, the invention features a compound having Formula
117:

[0497] In another embodiment, a compound having Formula 117 is used to
generate a compound having Formula 105 via phosphoramidite mediated
coupling with a biologically active molecule, such as a siNA molecule or
a portion thereof.

[0498] In one embodiment, the invention features a compound having Formula
118:

##STR00082##

[0499] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, R3
comprises H, OH, amino, substituted amino, nucleotide, nucleoside,
nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,
phospholipid, label, or a portion thereof, or OR5 where R5 a removable
protecting group, each R7 independently comprises an acyl group that can
be present or absent, for example a acetyl group, and each n is
independently an integer from about 1 to about 20. In another embodiment,
W is selected from the group consisting of amide, phosphate, phosphate
ester, phosphoramidate, or thiophosphate ester linkage.

[0500] In one embodiment, the invention features a compound having Formula
119:

##STR00083##

[0501] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, each
R7 independently comprises an acyl group that can be present or absent,
for example a acetyl group, and each n is independently an integer from
about 1 to about 20. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.

[0502] In one embodiment, the invention features a compound having Formula
120:

##STR00084##

[0503] wherein R3 comprises H, OH, amino, substituted amino, nucleotide,
nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,
lipid, phospholipid, label, or a portion thereof, or OR5 where R5 a
removable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,
O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7
independently comprises an acyl group that can be present or absent, for
example a acetyl group, each n is independently an integer from about 1
to about 20, and wherein R1 can include the groups:

##STR00085##

[0504] and wherein R2 can include the groups:

##STR00086##

[0505] In another embodiment, a compound having Formula 120 is used to
generate a compound having Formula 118 or 119 via phosphoramidite
mediated coupling with a biologically active molecule, such as a siNA
molecule or a portion thereof.

[0506] In one embodiment, the invention features a compound having Formula
121:

##STR00087##

[0507] wherein X comprises a siNA molecule or portion thereof; W comprises
a linker molecule or chemical linkage that can be present or absent, each
R7 independently comprises an acyl group that can be present or absent,
for example a acetyl group, and each n is independently an integer from
about 1 to about 20. In another embodiment, W is selected from the group
consisting of amide, phosphate, phosphate ester, phosphoramidate, or
thiophosphate ester linkage.

[0508] In one embodiment, the invention features a compound having Formula
122:

[0512] In another embodiment, a compound having Formula 122 is used to
generate a compound having Formula 121 via phosphoramidite mediated
coupling with a biologically active molecule, such as a siNA molecule or
a portion thereof.

[0513] In one embodiment, the invention features a compound having Formula
94,

[0515] In another embodiment, W of a compound having Formula 94 of the
invention comprises
5'-cytidine-deoxythymidine-3',5'-deoxythymidine-cytidine-3',5'-cytidine-d-
eoxyuridine-3',5'-deoxyuridine-cytidine-3',5'-uridine-deoxythymidine-3',
or 5'-deoxythymidine-uridine-3'.

[0516] In yet another embodiment, W of a compound having Formula 94 of the
invention comprises
5'-adenosine-deoxythymidine-3',5'-deoxythymidine-adenosine-3',5'-adenosin-
e-deoxyuridine-3', or 5'-deoxyuridine-adenosine-3'.

[0518] In another embodiment, compounds having Formula 89 and 91 of the
invention are synthesized by periodate oxidation of an N-terminal Serine
or Threonine residue of a peptide or protein.

[0519] In one embodiment, X of compounds having Formulae 43, 44, 46-52,
58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119,
or 121 of the invention comprises a siNA molecule or a portion thereof.
In one embodiment, the siNA molecule can be conjugated at the 5' end,
3'-end, or both 5' and 3' ends of the sense strand or region of the siNA.
In one embodiment, the siNA molecule can be conjugated at the 3'-end of
the antisense strand or region of the siNA with a compound of the
invention. In one embodiment, both the sense strand and antisense strands
or regions of the siNA molecule are conjugated with a compound of the
invention. In one embodiment, only the sense strand or region of the siNA
is conjugated with a compound of the invention. In one embodiment, only
the antisense strand or region of the siNA is conjugated with a compound
of the invention.

[0523] In one embodiment, the nucleic acid conjugates of the instant
invention are assembled by solid phase synthesis, for example on an
automated peptide synthesizer, for example a Miligen 9050 synthesizer
and/or an automated oligonucleotide synthesizer such as an ABI 394, 390Z,
or Pharmacia OligoProcess, OligoPilot, OligoMax, or AKTA synthesizer. In
another embodiment, the nucleic acid conjugates of the invention are
assembled post synthetically, for example, following solid phase
oligonucleotide synthesis (see for example FIGS. 45, 50, 53, and 73).

[0525] In one embodiment, the nucleic acid conjugates of the instant
invention are assembled post synthetically, for example, following solid
phase oligonucleotide synthesis.

[0526] The present invention provides compositions and conjugates
comprising nucleosidic and non-nucleosidic derivatives. The present
invention also provides nucleic acid, polynucleotide and oligonucleotide
derivatives including RNA, DNA, and PNA based conjugates. The attachment
of compounds of the invention to nucleosides, nucleotides,
non-nucleosides, and nucleic acid molecules is provided at any position
within the molecule, for example, at internucleotide linkages,
nucleosidic sugar hydroxyl groups such as 5', 3', and 2'-hydroxyls,
and/or at nucleobase positions such as amino and carbonyl groups.

[0527] The exemplary conjugates of the invention are described as
compounds of the formulae herein, however, other peptide, protein,
phospholipid, and poly-alkyl glycol derivatives are provided by the
invention, including various analogs of the compounds of formulae 1-122,
including but not limited to different isomers of the compounds described
herein.

[0528] The exemplary folate conjugates of the invention are described as
compounds shown by formulae herein, however, other folate and antifolate
derivatives are provided by the invention, including various folate
analogs of the formulae of the invention, including dihydrofloates,
tetrahydrofolates, tetrahydrorpterins, folinic acid, pteropolyglutamic
acid, 1-deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10
dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic
acids. As used herein, the term "folate" is meant to refer to folate and
folate derivatives, including pteroic acid derivatives and analogs.

[0529] The present invention features compositions and conjugates to
facilitate delivery of molecules into a biological system such as cells.
The conjugates provided by the instant invention can impart therapeutic
activity by transferring therapeutic compounds across cellular membranes.
The present invention encompasses the design and synthesis of novel
agents for the delivery of molecules, including but not limited to siNA
molecules. In general, the transporters described are designed to be used
either individually or as part of a multi-component system. The compounds
of the invention generally shown in Formulae herein are expected to
improve delivery of molecules into a number of cell types originating
from different tissues, in the presence or absence of serum.

[0530] In another embodiment, the compounds of the invention are provided
as a surface component of a lipid aggregate, such as a liposome
encapsulated with the predetermined molecule to be delivered. Liposomes,
which can be unilamellar or multilamellar, can introduce encapsulated
material into a cell by different mechanisms. For example, the liposome
can directly introduce its encapsulated material into the cell cytoplasm
by fusing with the cell membrane. Alternatively, the liposome can be
compartmentalized into an acidic vacuole (i.e., an endosome) and its
contents released from the liposome and out of the acidic vacuole into
the cellular cytoplasm.

[0531] In one embodiment the invention features a lipid aggregate
formulation of the compounds described herein, including
phosphatidylcholine (of varying chain length; e.g., egg yolk
phosphatidylcholine), cholesterol, a cationic lipid, and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polythyleneglycol-2000
(DSPE-PEG2000). The cationic lipid component of this lipid aggregate can
be any cationic lipid known in the art such as dioleoyl
1,2,-diacyl-3-trimethylammonium-propane (DOTAP). In another embodiment
this cationic lipid aggregate comprises a covalently bound compound
described in any of the Formulae herein.

[0532] In another embodiment, polyethylene glycol (PEG) is covalently
attached to the compounds of the present invention. The attached PEG can
be any molecular weight but is preferably between 2000-50,000 daltons.

[0533] The compounds and methods of the present invention are useful for
introducing nucleotides, nucleosides, nucleic acid molecules, lipids,
peptides, proteins, and/or non-nucleosidic small molecules into a cell.
For example, the invention can be used for nucleotide, nucleoside,
nucleic acid, lipids, peptides, proteins, and/or non-nucleosidic small
molecule delivery where the corresponding target site of action exists
intracellularly.

[0534] In one embodiment, the compounds of the instant invention provide
conjugates of molecules that can interact with cellular receptors, such
as high affinity folate receptors and ASGPr receptors, and provide a
number of features that allow the efficient delivery and subsequent
release of conjugated compounds across biological membranes. The
compounds utilize chemical linkages between the receptor ligand and the
compound to be delivered of length that can interact preferentially with
cellular receptors. Furthermore, the chemical linkages between the ligand
and the compound to be delivered can be designed as degradable linkages,
for example by utilizing a phosphate linkage that is proximal to a
nucleophile, such as a hydroxyl group. Deprotonation of the hydroxyl
group or an equivalent group, as a result of pH or interaction with a
nuclease, can result in nucleophilic attack of the phosphate resulting in
a cyclic phosphate intermediate that can be hydrolyzed. This cleavage
mechanism is analogous RNA cleavage in the presence of a base or RNA
nuclease. Alternately, other degradable linkages can be selected that
respond to various factors such as UV irradiation, cellular nucleases,
pH, temperature etc. The use of degradable linkages allows the delivered
compound to be released in a predetermined system, for example in the
cytoplasm of a cell, or in a particular cellular organelle.

[0535] The present invention also provides ligand derived phosphoramidites
that are readily conjugated to compounds and molecules of interest.
Phosphoramidite compounds of the invention permit the direct attachment
of conjugates to molecules of interest without the need for using nucleic
acid phosphoramidite species as scaffolds. As such, the used of
phosphoramidite chemistry can be used directly in coupling the compounds
of the invention to a compound of interest, without the need for other
condensation reactions, such as condensation of the ligand to an amino
group on the nucleic acid, for example at the N6 position of adenosine or
a 2'-deoxy-2'-amino function. Additionally, compounds of the invention
can be used to introduce non-nucleic acid based conjugated linkages into
oligonucleotides that can provide more efficient coupling during
oligonucleotide synthesis than the use of nucleic acid-based
phosphoramidites. This improved coupling can take into account improved
steric considerations of abasic or non-nucleosidic scaffolds bearing
pendant alkyl linkages.

[0537] The term "biodegradable linker" as used herein, refers to a nucleic
acid or non-nucleic acid linker molecule that is designed as a
biodegradable linker to connect one molecule to another molecule, for
example, a biologically active molecule to a siNA molecule of the
invention or the sense and antisense strands of a siNA molecule of the
invention. The biodegradable linker is designed such that its stability
can be modulated for a particular purpose, such as delivery to a
particular tissue or cell type. The stability of a nucleic acid-based
biodegradable linker molecule can be modulated by using various
chemistries, for example combinations of ribonucleotides,
deoxyribonucleotides, and chemically-modified nucleotides, such as
2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and
other 2'-modified or base modified nucleotides. The biodegradable nucleic
acid linker molecule can be a dimer, trimer, tetramer or longer nucleic
acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in
length, or can comprise a single nucleotide with a phosphorus-based
linkage, for example, a phosphoramidate or phosphodiester linkage. The
biodegradable nucleic acid linker molecule can also comprise nucleic acid
backbone, nucleic acid sugar, or nucleic acid base modifications.

[0538] The term "biodegradable" as used herein, refers to degradation in a
biological system, for example enzymatic degradation or chemical
degradation.

[0539] The term "biologically active molecule" as used herein, refers to
compounds or molecules that are capable of eliciting or modifying a
biological response in a system. Non-limiting examples of biologically
active siNA molecules either alone or in combination with other molecules
contemplated by the instant invention include therapeutically active
molecules such as antibodies, cholesterol, hormones, antivirals,
peptides, proteins, chemotherapeutics, small molecules, vitamins,
co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic
acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A
chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof.
Biologically active molecules of the invention also include molecules
capable of modulating the pharmacokinetics and/or pharmacodynamics of
other biologically active molecules, for example, lipids and polymers
such as polyamines, polyamides, polyethylene glycol and other polyethers.

[0540] The term "phospholipid" as used herein, refers to a hydrophobic
molecule comprising at least one phosphorus group. For example, a
phospholipid can comprise a phosphorus-containing group and saturated or
unsaturated alkyl group, optionally substituted with OH, COOH, oxo,
amine, or substituted or unsubstituted aryl groups.

[0541] The term "alkyl" as used herein refers to a saturated aliphatic
hydrocarbon, including straight-chain, branched-chain "isoalkyl", and
cyclic alkyl groups. The term "alkyl" also comprises alkoxy, alkyl-thio,
alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy,
cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,
C1-C6 hydrocarbyl, aryl or substituted aryl groups. Preferably, the alkyl
group has 1 to 12 carbons. More preferably it is a lower alkyl of from
about 1 to about 7 carbons, more preferably about 1 to about 4 carbons.
The alkyl group can be substituted or unsubstituted. When substituted the
substituted group(s) preferably comprise hydroxy, oxy, thio, amino,
nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl,
alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl,
cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or
substituted aryl groups. The term "alkyl" also includes alkenyl groups
containing at least one carbon-carbon double bond, including
straight-chain, branched-chain, and cyclic groups. Preferably, the
alkenyl group has about 2 to about 12 carbons. More preferably it is a
lower alkenyl of from about 2 to about 7 carbons, more preferably about 2
to about 4 carbons. The alkenyl group can be substituted or
unsubstituted. When substituted the substituted group(s) preferably
comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio,
alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl,
alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl,
heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups. The term
"alkyl" also includes alkynyl groups containing at least one
carbon-carbon triple bond, including straight-chain, branched-chain, and
cyclic groups. Preferably, the alkynyl group has about 2 to about 12
carbons. More preferably it is a lower alkynyl of from about 2 to about 7
carbons, more preferably about 2 to about 4 carbons. The alkynyl group
can be substituted or unsubstituted. When substituted the substituted
group(s) preferably comprise hydroxy, oxy, thio, amino, nitro, cyano,
alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl,
alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl,
heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl
groups. Alkyl groups or moieties of the invention can also include aryl,
alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.
The preferred substituent(s) of aryl groups are halogen, trihalomethyl,
hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino
groups. An "alkylaryl" group refers to an alkyl group (as described
above) covalently joined to an aryl group (as described above).
Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic
ring are all carbon atoms. The carbon atoms are optionally substituted.
Heterocyclic aryl groups are groups having from about 1 to about 3
heteroatoms as ring atoms in the aromatic ring and the remainder of the
ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur,
and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower
alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all
optionally substituted. An "amide" refers to an --C(O)--NH--R, where R is
either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an
--C(O)--OR', where R is either alkyl, aryl, alkylaryl or hydrogen.

[0542] The term "alkoxyalkyl" as used herein refers to an alkyl-O-alkyl
ether, for example, methoxyethyl or ethoxymethyl.

[0543] The term "alkyl-thio-alkyl" as used herein refers to an
alkyl-5-alkyl thioether, for example, methylthiomethyl or
methylthioethyl.

[0544] The term "amino" as used herein refers to a nitrogen containing
group as is known in the art derived from ammonia by the replacement of
one or more hydrogen radicals by organic radicals. For example, the terms
"aminoacyl" and "aminoalkyl" refer to specific N-substituted organic
radicals with acyl and alkyl substituent groups respectively.

[0545] The term "amination" as used herein refers to a process in which an
amino group or substituted amine is introduced into an organic molecule.

[0546] The term "exocyclic amine protecting moiety" as used herein refers
to a nucleobase amino protecting group compatible with oligonucleotide
synthesis, for example, an acyl or amide group.

[0547] The term "alkenyl" as used herein refers to a straight or branched
hydrocarbon of a designed number of carbon atoms containing at least one
carbon-carbon double bond. Examples of "alkenyl" include vinyl, allyl,
and 2-methyl-3-heptene.

[0548] The term "alkoxy" as used herein refers to an alkyl group of
indicated number of carbon atoms attached to the parent molecular moiety
through an oxygen bridge. Examples of alkoxy groups include, for example,
methoxy, ethoxy, propoxy and isopropoxy.

[0549] The term "alkynyl" as used herein refers to a straight or branched
hydrocarbon of a designed number of carbon atoms containing at least one
carbon-carbon triple bond. Examples of "alkynyl" include propargyl,
propyne, and 3-hexyne.

[0550] The term "aryl" as used herein refers to an aromatic hydrocarbon
ring system containing at least one aromatic ring. The aromatic ring can
optionally be fused or otherwise attached to other aromatic hydrocarbon
rings or non-aromatic hydrocarbon rings. Examples of aryl groups include,
for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene and
biphenyl. Preferred examples of aryl groups include phenyl and naphthyl.

[0551] The term "cycloalkenyl" as used herein refers to a C3-C8 cyclic
hydrocarbon containing at least one carbon-carbon double bond. Examples
of cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl,
cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,
cycloheptatrienyl, and cyclooctenyl.

[0552] The term "cycloalkyl" as used herein refers to a C3-C8 cyclic
hydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0553] The term "cycloalkylalkyl," as used herein, refers to a C3-C7
cycloalkyl group attached to the parent molecular moiety through an alkyl
group, as defined above. Examples of cycloalkylalkyl groups include
cyclopropylmethyl and cyclopentylethyl.

[0554] The terms "halogen" or "halo" as used herein refers to indicate
fluorine, chlorine, bromine, and iodine.

[0555] The term "heterocycloalkyl," as used herein refers to a
non-aromatic ring system containing at least one heteroatom selected from
nitrogen, oxygen, and sulfur. The heterocycloalkyl ring can be optionally
fused to or otherwise attached to other heterocycloalkyl rings and/or
non-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups have
from 3 to 7 members. Examples of heterocycloalkyl groups include, for
example, piperazine, morpholine, piperidine, tetrahydrofuran,
pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups include
piperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl.

[0556] The term "heteroaryl" as used herein refers to an aromatic ring
system containing at least one heteroatom selected from nitrogen, oxygen,
and sulfur. The heteroaryl ring can be fused or otherwise attached to one
or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings or
heterocycloalkyl rings. Examples of heteroaryl groups include, for
example, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline and
pyrimidine. Preferred examples of heteroaryl groups include thienyl,
benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl,
benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl,
isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl,
tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.

[0557] The term "C1-C6 hydrocarbyl" as used herein refers to straight,
branched, or cyclic alkyl groups having 1-6 carbon atoms, optionally
containing one or more carbon-carbon double or triple bonds. Examples of
hydrocarbyl groups include, for example, methyl, ethyl, propyl,
isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,
neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene,
cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl and
propargyl. When reference is made herein to C1-C6 hydrocarbyl containing
one or two double or triple bonds it is understood that at least two
carbons are present in the alkyl for one double or triple bond, and at
least four carbons for two double or triple bonds.

[0558] The term "protecting group" as used herein, refers to groups known
in the art that are readily introduced and removed from an atom, for
example O, N, P, or S. Protecting groups are used to prevent undesirable
reactions from taking place that can compete with the formation of a
specific compound or intermediate of interest. See also "Protective
Groups in Organic Synthesis", 3rd Ed., 1999, Greene, T. W. and related
publications.

[0559] The term "nitrogen protecting group," as used herein, refers to
groups known in the art that are readily introduced on to and removed
from a nitrogen. Examples of nitrogen protecting groups include Boc, Cbz,
benzoyl, and benzyl. See also "Protective Groups in Organic Synthesis",
3rd Ed., 1999, Greene, T. W. and related publications.

[0560] The term "hydroxy protecting group," or "hydroxy protection" as
used herein, refers to groups known in the art that are readily
introduced on to and removed from an oxygen, specifically an --OH group.
Examples of hyroxy protecting groups include trityl or substituted trityl
groups, such as monomethoxytrityl and dimethoxytrityl, or substituted
silyl groups, such as tert-butyldimethyl, trimethylsilyl, or
tert-butyldiphenyl silyl groups. See also "Protective Groups in Organic
Synthesis", 3rd Ed., 1999, Greene, T. W. and related publications.

[0561] The term "acyl" as used herein refers to --C(O)R groups, wherein R
is an alkyl or aryl.

[0562] The term "phosphorus containing group" as used herein, refers to a
chemical group containing a phosphorus atom. The phosphorus atom can be
trivalent or pentavalent, and can be substituted with O, H, N, S, C or
halogen atoms. Examples of phosphorus containing groups of the instant
invention include but are not limited to phosphorus atoms substituted
with O, H, N, S, C or halogen atoms, comprising phosphonate,
alkylphosphonate, phosphate, diphosphate, triphosphate, pyrophosphate,
phosphorothioate, phosphorodithioate, phosphoramidate, phosphoramidite
groups, nucleotides and nucleic acid molecules.

[0563] The term "phosphine" or "phosphite" as used herein refers to a
trivalent phosphorus species, for example compounds having Formula 97:

##STR00092## [0564] wherein R can include the groups:

[0564] ##STR00093## [0565] and wherein S and T independently include
the groups:

##STR00094##

[0566] The term "phosphate" as used herein refers to a pentavalent
phosphorus species, for example a compound having Formula 98:

##STR00095## [0567] wherein R includes the groups:

##STR00096##

[0568] and wherein S and T each independently can be a sulfur or oxygen
atom or a group which can include:

##STR00097##

[0569] and wherein M comprises a sulfur or oxygen atom. The phosphate of
the invention can comprise a nucleotide phosphate, wherein any R, S, or T
in Formula 98 comprises a linkage to a nucleic acid or nucleoside.

[0570] The term "cationic salt" as used herein refers to any organic or
inorganic salt having a net positive charge, for example a
triethylammonium (TEA) salt.

[0571] The term "degradable linker" as used herein, refers to linker
moieties that are capable of cleavage under various conditions.
Conditions suitable for cleavage can include but are not limited to pH,
UV irradiation, enzymatic activity, temperature, hydrolysis, elimination,
and substitution reactions, and thermodynamic properties of the linkage.

[0572] The term "photolabile linker" as used herein, refers to linker
moieties as are known in the art, that are selectively cleaved under
particular UV wavelengths. Compounds of the invention containing
photolabile linkers can be used to deliver compounds to a target cell or
tissue of interest, and can be subsequently released in the presence of a
UV source.

[0573] The term "nucleic acid conjugates" as used herein, refers to
nucleoside, nucleotide and oligonucleotide conjugates.

[0574] The term "lipid" as used herein, refers to any lipophilic compound.
Non-limiting examples of lipid compounds include fatty acids and their
derivatives, including straight chain, branched chain, saturated and
unsaturated fatty acids, carotenoids, terpenes, bile acids, and steroids,
including cholesterol and derivatives or analogs thereof.

[0576] The term "compounds with neutral charge" as used herein, refers to
compositions which are neutral or uncharged at neutral or physiological
pH. Examples of such compounds are cholesterol and other steroids,
cholesteryl hemisuccinate (CHEMS), dioleoyl phosphatidyl choline,
distearoylphosphotidyl choline (DSPC), fatty acids such as oleic acid,
phosphatidic acid and its derivatives, phosphatidyl serine, polyethylene
glycol-conjugated phosphatidylamine, phosphatidylcholine,
phosphatidylethanolamine and related variants, prenylated compounds
including farnesol, polyprenols, tocopherol, and their modified forms,
diacylsuccinyl glycerols, fusogenic or pore forming peptides,
dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.

[0577] The term "lipid aggregate" as used herein refers to a
lipid-containing composition wherein the lipid is in the form of a
liposome, micelle (non-lamellar phase) or other aggregates with one or
more lipids.

[0578] The term "nitrogen containing group" as used herein refers to any
chemical group or moiety comprising a nitrogen or substituted nitrogen.
Non-limiting examples of nitrogen containing groups include amines,
substituted amines, amides, alkylamines, amino acids such as arginine or
lysine, polyamines such as spermine or spermidine, cyclic amines such as
pyridines, pyrimidines including uracil, thymine, and cytosine,
morpholines, phthalimides, and heterocyclic amines such as purines,
including guanine and adenine.

[0579] Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered
exogenously optimally are stable within cells until reverse transcription
of the RNA has been modulated long enough to reduce the levels of the RNA
transcript. The nucleic acid molecules are resistant to nucleases in
order to function as effective intracellular therapeutic agents.
Improvements in the chemical synthesis of nucleic acid molecules
described in the instant invention and in the art have expanded the
ability to modify nucleic acid molecules by introducing nucleotide
modifications to enhance their nuclease stability as described above.

[0580] In yet another embodiment, siNA molecules having chemical
modifications that maintain or enhance enzymatic activity of proteins
involved in RNAi are provided. Such nucleic acids are also generally more
resistant to nucleases than unmodified nucleic acids. Thus, in vitro
and/or in vivo the activity should not be significantly lowered.

[0581] Use of the nucleic acid-based molecules of the invention will lead
to better treatment of the disease progression by affording the
possibility of combination therapies (e.g., multiple siNA molecules
targeted to different genes; nucleic acid molecules coupled with known
small molecule modulators; or intermittent treatment with combinations of
molecules, including different motifs and/or other chemical or biological
molecules). The treatment of subjects with siNA molecules can also
include combinations of different types of nucleic acid molecules, such
as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense,
2,5-A oligoadenylate, decoys, and aptamers.

[0582] In another aspect a siNA molecule of the invention comprises one or
more 5' and/or a 3'-cap structure, for example on only the sense siNA
strand, the antisense siNA strand, or both siNA strands.

[0585] By the term "non-nucleotide" is meant any group or compound which
can be incorporated into a nucleic acid chain in the place of one or more
nucleotide units, including either sugar and/or phosphate substitutions,
and allows the remaining bases to exhibit their enzymatic activity. The
group or compound is abasic in that it does not contain a commonly
recognized nucleotide base, such as adenosine, guanine, cytosine, uracil
or thymine and therefore lacks a base at the 1'-position.

[0586] By "nucleotide" as used herein is as recognized in the art to
include natural bases (standard), and modified bases well known in the
art. Such bases are generally located at the 1' position of a nucleotide
sugar moiety. Nucleotides generally comprise a base, sugar and a
phosphate group. The nucleotides can be unmodified or modified at the
sugar, phosphate and/or base moiety, (also referred to interchangeably as
nucleotide analogs, modified nucleotides, non-natural nucleotides,
non-standard nucleotides and other; see, for example, Usman and
McSwiggen, supra; Eckstein et al., International PCT Publication No. WO
92/07065; Usman et al., International PCT Publication No. WO 93/15187;
Uhlman & Peyman, supra, all are hereby incorporated by reference herein).
There are several examples of modified nucleic acid bases known in the
art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
Some of the non-limiting examples of base modifications that can be
introduced into nucleic acid molecules include, inosine, purine,
pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy
benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,
5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,
ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines
or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin
et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By
"modified bases" in this aspect is meant nucleotide bases other than
adenine, guanine, cytosine and uracil at 1' position or their
equivalents.

[0588] By "abasic" is meant sugar moieties lacking a base or having other
chemical groups in place of a base at the l' position, see for example
Adamic et al., U.S. Pat. No. 5,998,203.

[0589] By "unmodified nucleoside" is meant one of the bases adenine,
cytosine, guanine, thymine, or uracil joined to the 1' carbon of
β-D-ribo-furanose.

[0590] By "modified nucleoside" is meant any nucleotide base which
contains a modification in the chemical structure of an unmodified
nucleotide base, sugar and/or phosphate. Non-limiting examples of
modified nucleotides are shown by Formulae I-VII and/or other
modifications described herein.

[0591] In connection with 2'-modified nucleotides as described for the
present invention, by "amino" is meant 2'--NH2 or 2'-O--NH2,
which can be modified or unmodified. Such modified groups are described,
for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and
Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both
incorporated by reference in their entireties.

[0592] Various modifications to nucleic acid siNA structure can be made to
enhance the utility of these molecules. Such modifications will enhance
shelf-life, half-life in vitro, stability, and ease of introduction of
such oligonucleotides to the target site, e.g., to enhance penetration of
cellular membranes, and confer the ability to recognize and bind to
targeted cells.

Administration of Nucleic Acid Molecules

[0593] A siNA molecule of the invention can be adapted for use to treat
any disease, infection or condition associated with gene expression, and
other indications that can respond to the level of gene product in a cell
or tissue, alone or in combination with other therapies. For example, a
siNA molecule can comprise a delivery vehicle, including liposomes, for
administration to a subject, carriers and diluents and their salts,
and/or can be present in pharmaceutically acceptable formulations.
Methods for the delivery of nucleic acid molecules are described in
Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for
Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al.,
1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb.
Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752,
184-192, all of which are incorporated herein by reference. Beigelman et
al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further
describe the general methods for delivery of nucleic acid molecules.
These protocols can be utilized for the delivery of virtually any nucleic
acid molecule. Nucleic acid molecules can be administered to cells by a
variety of methods known to those of skill in the art, including, but not
restricted to, encapsulation in liposomes, by iontophoresis, or by
incorporation into other vehicles, such as biodegradable polymers,
hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,
Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT
publication Nos. WO 03/47518 and WO 03/46185),
poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for
example U.S. Pat. No. 6,447,796 and US Patent Application Publication No.
US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres,
or by proteinaceous vectors (O'Hare and Normand, International PCT
Publication No. WO 00/53722). In one embodiment, nucleic acid molecules
or the invention are administered via biodegradable implant materials,
such as elastic shape memory polymers (see for example Lendelein and
Langer, 2002, Science, 296, 1673). Alternatively, the nucleic
acid/vehicle combination is locally delivered by direct injection or by
use of an infusion pump. Direct injection of the nucleic acid molecules
of the invention, whether subcutaneous, intramuscular, or intradermal,
can take place using standard needle and syringe methodologies, or by
needle-free technologies such as those described in Conry et al., 1999,
Clin. Cancer Res., 5, 2330-2337 and Barry et al., International PCT
Publication No. WO 99/31262. Many examples in the art describe CNS
delivery methods of oligonucleotides by osmotic pump, (see Chun et al.,
1998, Neuroscience Letters, 257, 135-138, D'Aldin et al., 1998, Mol.
Brain Research, 55, 151-164, Dryden et al., 1998, J. Endocrinol., 157,
169-175, Ghirnikar et al., 1998, Neuroscience Letters, 247, 21-24) or
direct infusion (Broaddus et al., 1997, Neurosurg. Focus, 3, article 4).
Other routes of delivery include, but are not limited to oral (tablet or
pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76,
1153-1158). More detailed descriptions of nucleic acid delivery and
administration are provided in Sullivan et al., supra, Draper et al., PCT
WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT
WO99/04819 all of which have been incorporated by reference herein. The
molecules of the instant invention can be used as pharmaceutical agents.
Pharmaceutical agents prevent, modulate the occurrence, or treat
(alleviate a symptom to some extent, preferably all of the symptoms) of a
disease state in a subject.

[0594] In addition, the invention features the use of methods to deliver
the nucleic acid molecules of the instant invention to hematopoietic
cells, including monocytes and lymphocytes. These methods are described
in detail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2),
920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion and
Phillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,
1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, Nucleic
Acids Research, 22(22), 4681-8. Such methods, as described above, include
the use of free oligonucleotide, cationic lipid formulations, liposome
formulations including pH sensitive liposomes and immunoliposomes, and
bioconjugates including oligonucleotides conjugated to fusogenic
peptides, for the transfection of hematopoietic cells with
oligonucleotides.

[0595] In one embodiment, a compound, molecule, or composition for the
treatment of ocular conditions (e.g., macular degeneration, diabetic
retinopathy etc.) is administered to a subject intraocularly or by
intraocular means. In another embodiment, a compound, molecule, or
composition for the treatment of ocular conditions (e.g., macular
degeneration, diabetic retinopathy etc.) is administered to a subject
periocularly or by periocular means (see for example Ahlheim et al.,
International PCT publication No. WO 03/24420). In one embodiment, a siNA
molecule and/or formulation or composition thereof is administered to a
subject intraocularly or by intraocular means. In another embodiment, a
siNA molecule and/or formulation or composition thereof is administered
to a subject periocularly or by periocular means. Periocular
administration generally provides a less invasive approach to
administering siNA molecules and formulation or composition thereof to a
subject (see for example Ahlheim et al., International PCT publication
No. WO 03/24420). The use of periocular administraction also minimizes
the risk of retinal detachment, allows for more frequent dosing or
administraction, provides a clinically relevant route of administraction
for macular degeneration and other optic conditions, and also provides
the possibility of using resevoirs (e.g., implants, pumps or other
devices) for drug delivery.

[0596] In one embodiment, a siNA molecule of the invention is complexed
with membrane disruptive agents such as those described in U.S. Patent
Appliaction Publication No. 20010007666, incorporated by reference herein
in its entirety including the drawings. In another embodiment, the
membrane disruptive agent or agents and the siNA molecule are also
complexed with a cationic lipid or helper lipid molecule, such as those
lipids described in U.S. Pat. No. 6,235,310, incorporated by reference
herein in its entirety including the drawings.

[0598] In one embodiment, a siNA molecule of the invention comprises a
bioconjugate, for example a nucleic acid conjugate as described in
Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S. Pat.
No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886; U.S.
Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,138,045, all
incorporated by reference herein.

[0599] Thus, the invention features a pharmaceutical composition
comprising one or more nucleic acid(s) of the invention in an acceptable
carrier, such as a stabilizer, buffer, and the like. The polynucleotides
of the invention can be administered (e.g., RNA, DNA or protein) and
introduced into a subject by any standard means, with or without
stabilizers, buffers, and the like, to form a pharmaceutical composition.
When it is desired to use a liposome delivery mechanism, standard
protocols for formation of liposomes can be followed. The compositions of
the present invention can also be formulated and used as tablets,
capsules or elixirs for oral administration, suppositories for rectal
administration, sterile solutions, suspensions for injectable
administration, and the other compositions known in the art.

[0600] The present invention also includes pharmaceutically acceptable
formulations of the compounds described. These formulations include salts
of the above compounds, e.g., acid addition salts, for example, salts of
hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

[0601] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration, e.g.,
systemic administration, into a cell or subject, including for example a
human. Suitable forms, in part, depend upon the use or the route of
entry, for example oral, transdermal, or by injection. Such forms should
not prevent the composition or formulation from reaching a target cell
(i.e., a cell to which the negatively charged nucleic acid is desirable
for delivery). For example, pharmacological compositions injected into
the blood stream should be soluble. Other factors are known in the art,
and include considerations such as toxicity and forms that prevent the
composition or formulation from exerting its effect.

[0602] By "systemic administration" is meant in vivo systemic absorption
or accumulation of drugs in the blood stream followed by distribution
throughout the entire body. Administration routes that lead to systemic
absorption include, without limitation: intravenous, subcutaneous,
intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each
of these administration routes exposes the siNA molecules of the
invention to an accessible diseased tissue. The rate of entry of a drug
into the circulation has been shown to be a function of molecular weight
or size. The use of a liposome or other drug carrier comprising the
compounds of the instant invention can potentially localize the drug, for
example, in certain tissue types, such as the tissues of the reticular
endothelial system (RES). A liposome formulation that can facilitate the
association of drug with the surface of cells, such as, lymphocytes and
macrophages is also useful. This approach can provide enhanced delivery
of the drug to target cells by taking advantage of the specificity of
macrophage and lymphocyte immune recognition of abnormal cells, such as
cancer cells.

[0605] The present invention also includes compositions prepared for
storage or administration that include a pharmaceutically effective
amount of the desired compounds in a pharmaceutically acceptable carrier
or diluent. Acceptable carriers or diluents for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro
edit. 1985), hereby incorporated by reference herein. For example,
preservatives, stabilizers, dyes and flavoring agents can be provided.
These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic
acid. In addition, antioxidants and suspending agents can be used.

[0606] A pharmaceutically effective dose is that dose required to prevent,
inhibit the occurrence, or treat (alleviate a symptom to some extent,
preferably all of the symptoms) of a disease state. The pharmaceutically
effective dose depends on the type of disease, the composition used, the
route of administration, the type of mammal being treated, the physical
characteristics of the specific mammal under consideration, concurrent
medication, and other factors that those skilled in the medical arts will
recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body
weight/day of active ingredients is administered dependent upon potency
of the negatively charged polymer.

[0607] The nucleic acid molecules of the invention and formulations
thereof can be administered orally, topically, parenterally, by
inhalation or spray, or rectally in dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers, adjuvants
and/or vehicles. The term parenteral as used herein includes
percutaneous, subcutaneous, intravascular (e.g., intravenous),
intramuscular, or intrathecal injection or infusion techniques and the
like. In addition, there is provided a pharmaceutical formulation
comprising a nucleic acid molecule of the invention and a
pharmaceutically acceptable carrier. One or more nucleic acid molecules
of the invention can be present in association with one or more non-toxic
pharmaceutically acceptable carriers and/or diluents and/or adjuvants,
and if desired other active ingredients. The pharmaceutical compositions
containing nucleic acid molecules of the invention can be in a form
suitable for oral use, for example, as tablets, troches, lozenges,
aqueous or oily suspensions, dispersible powders or granules, emulsion,
hard or soft capsules, or syrups or elixirs.

[0608] Compositions intended for oral use can be prepared according to any
method known to the art for the manufacture of pharmaceutical
compositions and such compositions can contain one or more such
sweetening agents, flavoring agents, coloring agents or preservative
agents in order to provide pharmaceutically elegant and palatable
preparations. Tablets contain the active ingredient in admixture with
non-toxic pharmaceutically acceptable excipients that are suitable for
the manufacture of tablets. These excipients can be, for example, inert
diluents; such as calcium carbonate, sodium carbonate, lactose, calcium
phosphate or sodium phosphate; granulating and disintegrating agents, for
example, corn starch, or alginic acid; binding agents, for example
starch, gelatin or acacia; and lubricating agents, for example magnesium
stearate, stearic acid or talc. The tablets can be uncoated or they can
be coated by known techniques. In some cases such coatings can be
prepared by known techniques to delay disintegration and absorption in
the gastrointestinal tract and thereby provide a sustained action over a
longer period. For example, a time delay material such as glyceryl
monosterate or glyceryl distearate can be employed.

[0609] Formulations for oral use can also be presented as hard gelatin
capsules wherein the active ingredient is mixed with an inert solid
diluent, for example, calcium carbonate, calcium phosphate or kaolin, or
as soft gelatin capsules wherein the active ingredient is mixed with
water or an oil medium, for example peanut oil, liquid paraffin or olive
oil.

[0610] Aqueous suspensions contain the active materials in a mixture with
excipients suitable for the manufacture of aqueous suspensions. Such
excipients are suspending agents, for example sodium
carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,
sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;
dispersing or wetting agents can be a naturally-occurring phosphatide,
for example, lecithin, or condensation products of an alkylene oxide with
fatty acids, for example polyoxyethylene stearate, or condensation
products of ethylene oxide with long chain aliphatic alcohols, for
example heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol such as
polyoxyethylene sorbitol monooleate, or condensation products of ethylene
oxide with partial esters derived from fatty acids and hexitol
anhydrides, for example polyethylene sorbitan monooleate. The aqueous
suspensions can also contain one or more preservatives, for example
ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or
more flavoring agents, and one or more sweetening agents, such as sucrose
or saccharin.

[0611] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
The oily suspensions can contain a thickening agent, for example beeswax,
hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents
can be added to provide palatable oral preparations. These compositions
can be preserved by the addition of an anti-oxidant such as ascorbic acid

[0612] Dispersible powders and granules suitable for preparation of an
aqueous suspension by the addition of water provide the active ingredient
in admixture with a dispersing or wetting agent, suspending agent and one
or more preservatives. Suitable dispersing or wetting agents or
suspending agents are exemplified by those already mentioned above.
Additional excipients, for example sweetening, flavoring and coloring
agents, can also be present.

[0613] Pharmaceutical compositions of the invention can also be in the
form of oil-in-water emulsions. The oily phase can be a vegetable oil or
a mineral oil or mixtures of these. Suitable emulsifying agents can be
naturally-occurring gums, for example gum acacia or gum tragacanth,
naturally-occurring phosphatides, for example soy bean, lecithin, and
esters or partial esters derived from fatty acids and hexitol,
anhydrides, for example sorbitan monooleate, and condensation products of
the said partial esters with ethylene oxide, for example polyoxyethylene
sorbitan monooleate. The emulsions can also contain sweetening and
flavoring agents.

[0614] Syrups and elixirs can be formulated with sweetening agents, for
example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such
formulations can also contain a demulcent, a preservative and flavoring
and coloring agents. The pharmaceutical compositions can be in the form
of a sterile injectable aqueous or oleaginous suspension. This suspension
can be formulated according to the known art using those suitable
dispersing or wetting agents and suspending agents that have been
mentioned above. The sterile injectable preparation can also be a sterile
injectable solution or suspension in a non-toxic parentally acceptable
diluent or solvent, for example as a solution in 1,3-butanediol. Among
the acceptable vehicles and solvents that can be employed are water,
Ringer's solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil can be employed
including synthetic mono- or diglycerides. In addition, fatty acids such
as oleic acid find use in the preparation of injectables.

[0615] The nucleic acid molecules of the invention can also be
administered in the form of suppositories, e.g., for rectal
administration of the drug. These compositions can be prepared by mixing
the drug with a suitable non-irritating excipient that is solid at
ordinary temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials include
cocoa butter and polyethylene glycols.

[0616] Nucleic acid molecules of the invention can be administered
parenterally in a sterile medium. The drug, depending on the vehicle and
concentration used, can either be suspended or dissolved in the vehicle.
Advantageously, adjuvants such as local anesthetics, preservatives and
buffering agents can be dissolved in the vehicle.

[0617] Dosage levels of the order of from about 0.1 mg to about 140 mg per
kilogram of body weight per day are useful in the treatment of the
above-indicated conditions (about 0.5 mg to about 7 g per subject per
day). The amount of active ingredient that can be combined with the
carrier materials to produce a single dosage form varies depending upon
the host treated and the particular mode of administration. Dosage unit
forms generally contain between from about 1 mg to about 500 mg of an
active ingredient.

[0618] It is understood that the specific dose level for any particular
subject depends upon a variety of factors including the activity of the
specific compound employed, the age, body weight, general health, sex,
diet, time of administration, route of administration, and rate of
excretion, drug combination and the severity of the particular disease
undergoing therapy.

[0619] For administration to non-human animals, the composition can also
be added to the animal feed or drinking water. It can be convenient to
formulate the animal feed and drinking water compositions so that the
animal takes in a therapeutically appropriate quantity of the composition
along with its diet. It can also be convenient to present the composition
as a premix for addition to the feed or drinking water.

[0620] The nucleic acid molecules of the present invention can also be
administered to a subject in combination with other therapeutic compounds
to increase the overall therapeutic effect. The use of multiple compounds
to treat an indication can increase the beneficial effects while reducing
the presence of side effects.

[0621] In one embodiment, the invention comprises compositions suitable
for administering nucleic acid molecules of the invention to specific
cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and
Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and
binds branched galactose-terminal glycoproteins, such as
asialoorosomucoid (ASOR). In another example, the folate receptor is
overexpressed in many cancer cells. Binding of such glycoproteins,
synthetic glycoconjugates, or folates to the receptor takes place with an
affinity that strongly depends on the degree of branching of the
oligosaccharide chain, for example, triatennary structures are bound with
greater affinity than biatenarry or monoatennary chains (Baenziger and
Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem.,
257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained
this high specificity through the use of N-acetyl-D-galactosamine as the
carbohydrate moiety, which has higher affinity for the receptor, compared
to galactose. This "clustering effect" has also been described for the
binding and uptake of mannosyl-terminating glycoproteins or
glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395).
The use of galactose, galactosamine, or folate based conjugates to
transport exogenous compounds across cell membranes can provide a
targeted delivery approach to, for example, the treatment of liver
disease, cancers of the liver, or other cancers. The use of bioconjugates
can also provide a reduction in the required dose of therapeutic
compounds required for treatment. Furthermore, therapeutic
bioavialability, pharmacodynamics, and pharmacokinetic parameters can be
modulated through the use of nucleic acid bioconjugates of the invention.
Non-limiting examples of such bioconjugates are described in Vargeese et
al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et
al., U.S. Ser. No. 10/151,116, filed May 17, 2002. In one embodiment,
nucleic acid molecules of the invention are complexed with or covalently
attached to nanoparticles, such as Hepatitis B virus S, M, or L evelope
proteins (see for example Yamado et al., 2003, Nature Biotechnology, 21,
885). In one embodiment, nucleic acid molecules of the invention are
delivered with specificity for human tumor cells, specifically
non-apoptotic human tumor cells including for example T-cells,
hepatocytes, breast carcinoma cells, ovarian carcinoma cells, melanoma
cells, intestinal epithelial cells, prostate cells, testicular cells,
non-small cell lung cancers, small cell lung cancers, etc.

EXAMPLES

[0622] The following are non-limiting examples showing the selection,
isolation, synthesis and activity of nucleic acids of the instant
invention.

Example 1

Tandem Synthesis of siNA Constructs

[0623] Exemplary siNA molecules of the invention are synthesized in tandem
using a cleavable linker, for example, a succinyl-based linker. Tandem
synthesis as described herein is followed by a one-step purification
process that provides RNAi molecules in high yield. This approach is
highly amenable to siNA synthesis in support of high throughput RNAi
screening, and can be readily adapted to multi-column or multi-well
synthesis platforms.

[0624] After completing a tandem synthesis of a siNA oligo and its
complement in which the 5'-terminal dimethoxytrityl (5'-O-DMT) group
remains intact (trityl on synthesis), the oligonucleotides are
deprotected as described above. Following deprotection, the siNA sequence
strands are allowed to spontaneously hybridize. This hybridization yields
a duplex in which one strand has retained the 5'-O-DMT group while the
complementary strand comprises a terminal 5'-hydroxyl. The newly formed
duplex behaves as a single molecule during routine solid-phase extraction
purification (Trityl-On purification) even though only one molecule has a
dimethoxytrityl group. Because the strands form a stable duplex, this
dimethoxytrityl group (or an equivalent group, such as other trityl
groups or other hydrophobic moieties) is all that is required to purify
the pair of oligos, for example, by using a C18 cartridge.

[0625] Standard phosphoramidite synthesis chemistry is used up to the
point of introducing a tandem linker, such as an inverted deoxy abasic
succinate or glyceryl succinate linker (see FIG. 1) or an equivalent
cleavable linker. A non-limiting example of linker coupling conditions
that can be used includes a hindered base such as diisopropylethylamine
(DIPA) and/or DMAP in the presence of an activator reagent such as
Bromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After the
linker is coupled, standard synthesis chemistry is utilized to complete
synthesis of the second sequence leaving the terminal the 5'-O-DMT
intact. Following synthesis, the resulting oligonucleotide is deprotected
according to the procedures described herein and quenched with a suitable
buffer, for example with 50 mM NaOAc or 1.5M NH4H2CO3.

[0626] Purification of the siNA duplex can be readily accomplished using
solid phase extraction, for example using a Waters C18 SepPak 1 g
cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV
H2O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with
1 CV H2O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14%
ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed,
for example with 1 CV H2O followed by on-column detritylation, for
example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the
column, then adding a second CV of 1% aqueous TFA to the column and
allowing to stand for approximately 10 minutes. The remaining TFA
solution is removed and the column washed with H2O followed by 1 CV
1M NaCl and additional H2O. The siNA duplex product is then eluted,
for example, using 1 CV 20% aqueous CAN.

[0627] FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis
of a purified siNA construct in which each peak corresponds to the
calculated mass of an individual siNA strand of the siNA duplex. The same
purified siNA provides three peaks when analyzed by capillary gel
electrophoresis (CGE), one peak presumably corresponding to the duplex
siNA, and two peaks presumably corresponding to the separate siNA
sequence strands. Ion exchange HPLC analysis of the same siNA contract
only shows a single peak. Testing of the purified siNA construct using a
luciferase reporter assay described below demonstrated the same RNAi
activity compared to siNA constructs generated from separately
synthesized oligonucleotide sequence strands.

Example 2

Serum Stability of Chemically Modified siNA Constructs

[0628] Chemical modifications were introduced into siNA constructs to
determine the stability of these constructs compared to native siNA
oligonucleotides (containing two thymidine nucleotide overhangs) in human
serum. An investigation of the serum stability of RNA duplexes revealed
that siNA constructs consisting of all RNA nucleotides containing two
thymidine nucleotide overhangs have a half-life in serum of 15 seconds,
whereas chemically modified siNA constructs remained stable in serum for
1 to 3 days depending on the extent of modification (see FIG. 3). RNAi
stability tests were performed by internally labeling one strand (strand
1) of siNA and duplexing with 1.5× the concentration of the
complementary siNA strand (strand 2) (to insure all labeled material was
in duplex form). Duplexed siNA constructs were then tested for stability
by incubating at a final concentration of 2 μM siNA (strand 2
concentration) in 90% mouse or human serum for time-points of 30 sec, 1
min, 5 min, 30 min, 90 min, 4 hrs 10 min, 16 hrs 24 min, and 49 hrs. Time
points were run on a 15% denaturing polyacrylamide gels and analyzed on a
phosphoimager.

[0629] Internal labeling was performed via kinase reactions with
polynucleotide kinase (PNK) and 32P-γ-ATP, with addition of
radiolabeled phosphate at nucleotide 13 of strand 2, counting in from the
3' side. Ligation of the remaining 8-mer fragments with T4 RNA ligase
resulted in the full length, 21-mer, strand 2. Duplexing of RNAi was done
by adding appropriate concentrations of the siNA oligonucleotides and
heating to 95° C. for 5 minutes followed by slow cooling to room
temperature. Reactions were performed by adding 100% serum to the siNA
duplexes and incubating at 37° C., then removing aliquots at
desired time-points. Results of this study are summarized in FIG. 3. As
shown in the FIG. 3, chemically modified siNA molecules (e.g., SEQ ID
NOs: 412/413, 412/414, 412/415, 412/416, and 412/418) have significantly
increased serum stability compared to an siNA construct having all
ribonucleotides except a 3'-terminal dithymidine (TT) modification (e.g.,
SEQ ID NOs: 419/420).

Example 3

Identification of Potential siNA Target Sites in any RNA Sequence

[0630] The sequence of an RNA target of interest, such as a viral or human
mRNA transcript, is screened for target sites, for example by using a
computer folding algorithm. In a non-limiting example, the sequence of a
gene or RNA gene transcript derived from a database, such as Genbank, is
used to generate siNA targets having complementarity to the target. Such
sequences can be obtained from a database, or can be determined
experimentally as known in the art. Target sites that are known, for
example, those target sites determined to be effective target sites based
on studies with other nucleic acid molecules, for example ribozymes or
antisense, or those targets known to be associated with a disease or
condition such as those sites containing mutations or deletions, can be
used to design siNA molecules targeting those sites. Various parameters
can be used to determine which sites are the most suitable target sites
within the target RNA sequence. These parameters include but are not
limited to secondary or tertiary RNA structure, the nucleotide base
composition of the target sequence, the degree of homology between
various regions of the target sequence, or the relative position of the
target sequence within the RNA transcript. Based on these determinations,
any number of target sites within the RNA transcript can be chosen to
screen siNA molecules for efficacy, for example by using in vitro RNA
cleavage assays, cell culture, or animal models. In a non-limiting
example, anywhere from 1 to 1000 target sites are chosen within the
transcript based on the size of the siNA construct to be used. High
throughput screening assays can be developed for screening siNA molecules
using methods known in the art, such as with multi-well or multi-plate
assays or combinatorial/siNA library screening assays to determine
efficient reduction in target gene expression.

Example 4

Selection of siNA Molecule Target Sites in a RNA

[0631] The following non-limiting steps can be used to carry out the
selection of siNAs targeting a given gene sequence or transcript.

[0632] The target sequence is parsed in silico into a list of all
fragments or subsequences of a particular length, for example 23
nucleotide fragments, contained within the target sequence. This step is
typically carried out using a custom Perl script, but commercial sequence
analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package
can be employed as well.

[0633] In some instances the siNAs correspond to more than one target
sequence; such would be the case for example in targeting different
transcripts of the same gene, targeting different transcripts of more
than one gene, or for targeting both the human gene and an animal
homolog. In this case, a subsequence list of a particular length is
generated for each of the targets, and then the lists are compared to
find matching sequences in each list. The subsequences are then ranked
according to the number of target sequences that contain the given
subsequence; the goal is to find subsequences that are present in most or
all of the target sequences. Alternately, the ranking can identify
subsequences that are unique to a target sequence, such as a mutant
target sequence. Such an approach would enable the use of siNA to target
specifically the mutant sequence and not effect the expression of the
normal sequence.

[0634] In some instances the siNA subsequences are absent in one or more
sequences while present in the desired target sequence; such would be the
case if the siNA targets a gene with a paralogous family member that is
to remain untargeted. As in case 2 above, a subsequence list of a
particular length is generated for each of the targets, and then the
lists are compared to find sequences that are present in the target gene
but are absent in the untargeted paralog.

[0635] The ranked siNA subsequences can be further analyzed and ranked
according to GC content. A preference can be given to sites containing
30-70% GC, with a further preference to sites containing 40-60% GC.

[0636] The ranked siNA subsequences can be further analyzed and ranked
according to self-folding and internal hairpins. Weaker internal folds
are preferred; strong hairpin structures are to be avoided.

[0637] The ranked siNA subsequences can be further analyzed and ranked
according to whether they have runs of GGG or CCC in the sequence. GGG
(or even more Gs) in either strand can make oligonucleotide synthesis
problematic and can potentially interfere with RNAi activity, so it is
avoided other appropriately suitable sequences are available. CCC is
searched in the target strand because that will place GGG in the
antisense strand.

[0638] The ranked siNA subsequences can be further analyzed and ranked
according to whether they have the dinucleotide UU (uridine dinucleotide)
on the 3'-end of the sequence, and/or AA on the 5'-end of the sequence
(to yield 3' UU on the antisense sequence). These sequences allow one to
design siNA molecules with terminal TT thymidine dinucleotides.

[0639] Four or five target sites are chosen from the ranked list of
subsequences as described above. For example, in subsequences having 23
nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence
are then designed and synthesized for the upper (sense) strand of the
siNA duplex, while the reverse complement of the left 21 nucleotides of
each chosen 23-mer subsequence are then designed and synthesized for the
lower (antisense) strand of the siNA duplex (see Tables I). If terminal
TT residues are desired for the sequence (as described in paragraph 7),
then the two 3' terminal nucleotides of both the sense and antisense
strands are replaced by TT prior to synthesizing the oligos.

[0640] The siNA molecules are screened in an in vitro, cell culture or
animal model system to identify the most active siNA molecule or the most
preferred target site within the target RNA sequence.

[0641] In an alternate approach, a pool of siNA constructs specific to a
target sequence is used to screen for target sites in cells expressing
target RNA, such as human HeLa cells. The general strategy used in this
approach is shown in FIG. 21. A non-limiting example of such a pool is a
pool comprising sequences having antisense sequences complementary to the
target RNA sequence and sense sequences complementary to the antisense
sequences. Cells (e.g., HeLa cells) expressing the target gene are
transfected with the pool of siNA constructs and cells that demonstrate a
phenotype associated with gene silencing are sorted. The pool of siNA
constructs can be chemically modified as described herein and
synthesized, for example, in a high throughput manner. The siNA from
cells demonstrating a positive phenotypic change (e.g., decreased target
mRNA levels or target protein expression), are identified, for example by
positional analysis within the assay, and are used to determine the most
suitable target site(s) within the target RNA sequence based upon the
complementary sequence to the corresponding siNA antisense strand
identified in the assay.

Example 5

RNAi Activity of Chemically Modified siNA Constructs

[0642] Short interfering nucleic acid (siNA) is emerging as a powerful
tool for gene regulation. All-ribose siNA duplexes activate the RNAi
pathway but have limited utility as therapeutic compounds due to their
nuclease sensitivity and short half-life in serum, as shown in Example 2
above. To develop nuclease-resistant siNA constructs for in vivo
applications, siNAs that target luciferase mRNA and contain stabilizing
chemical modifications were tested for activity in HeLa cells. The
sequences for the siNA oligonucleotide sequences used in this study are
shown in Table I. Modifications included phosphorothioate linkages
(P═S), 2'-O-methyl nucleotides, or 2'-fluoro (F) nucleotides in one
or both siNA strands and various 3'-end stabilization chemistries,
including 3'-glyceryl, 3'-inverted abasic, 3'-inverted Thymidine, and/or
Thymidine. The RNAi activity of chemically stabilized siNA constructs was
compared with the RNAi activity of control siNA constructs consisting of
all ribonucleotides at every position except the 3'-terminus which
comprised two thymidine nucleotide overhangs. Active siNA molecules
containing stabilizing modifications such as described herein should
prove useful for in vivo applications, given their enhanced
nuclease-resistance.

[0644] HeLa S3 cells were grown at 37° C. in DMEM with 5% FBS and
seeded at 15,300 cells in 100 ul media per well of a 96-well plate 24
hours prior to transfection. For transfection, 4 ul Lipofectamine 2000
(Life Technologies) was added to 96 ul OPTI-MEM, vortexed and incubated
at room temperature for 5 minutes. The 100 ul diluted lipid was then
added to a microtiter tube containing 5 ul pGL2 (200 ng/u1), 5 ul pRLSV40
(8 ng/ul) 6 ul siNA (25 nM or 10 nM final), and 84 ul OPTI-MEM, vortexed
briefly and incubated at room temperature for 20 minutes. The
transfection mix was then mixed briefly and 50 ul was added to each of
three wells that contained HeLa S3 cells in 100 ul media. Cells were
incubated for 20 hours after transfection and analyzed for luciferase
expression using the Dual luciferase assay according to the
manufacturer's instructions (Promega Biotech). The results of this study
are summarized in FIGS. 4-16. The sequences of the siNA strands used in
this study are shown in Table I and are referred to by Sirna/RPI # in the
figures. Normalized luciferase activity is reported as the ratio of
firefly luciferase activity to renilla luciferase activity in the same
sample. Error bars represent standard deviation of triplicate
transfections. As shown in FIGS. 4-16, the RNAi activity of chemically
modified constructs is often comparable to that of unmodified control
siNA constructs, which consist of all ribonucleotides at every position
except the 3'-terminus which comprises two thymidine nucleotide
overhangs. In some instances, the RNAi activity of the chemically
modified constructs is greater than the unmodified control siNA construct
consisting of all ribonucleotides.

[0645] For example, FIG. 4 shows results obtained from a screen using
phosphorothioate modified siNA constructs. The Sirna/RPI 27654/27659
construct contains phosphorothioate substitutions for every pyrimidine
nucleotide in both sequences, the Sirna/RPI 27657/27662 construct
contains 5 terminal 3'-phosphorothioate substitutions in each strand, the
Sirna/RPI 27649/27658 construct contains all phosphorothioate
substitutions only in the antisense strand, whereas the Sirna/RPI
27649/27660 and Sirna/RPI 27649/27661 constructs have unmodified sense
strands and varying degrees of phosphorothioate substitutions in the
antisense strand. All of these constructs show significant RNAi activity
when compared to a scrambled siNA control construct (27651/27652).

[0646] FIG. 5 shows results obtained from a screen using phosphorothioate
(Sirna/RPI 28253/28255 and Sirna/RPI 28254/28256) and universal base
substitutions (Sirna/RPI 28257/28259 and Sirna/RPI 28258/28260) compared
to the same controls described above, these modifications show equivalent
or better RNAi activity when compared to the unmodified control siNA
construct.

[0647] FIG. 6 shows results obtained from a screen using 2'-O-methyl
modified siNA constructs in which the sense strand contains either 10
(Sirna/RPI 28244/27650) or 5 (Sirna/RPI 28245/27650) 2'-O-methyl
substitutions, both with comparable activity to the unmodified control
siNA construct.

[0648] FIG. 7 shows results obtained from a screen using 2'-O-methyl or
2'-deoxy-2'-fluoro modified siNA constructs compared to a control
construct consisting of all ribonucleotides at every position except the
3'-terminus which comprises two thymidine nucleotide overhangs.

[0649] FIG. 8 compares a siNA construct containing six phosphorothioate
substitutions in each strand (Sirna/RPI 28460/28461), where 5
phosphorothioates are present at the 3' end and a single phosphorothioate
is present at the 5' end of each strand. This motif shows very similar
activity to the control siNA construct consisting of all ribonucleotides
at every position except the 3'-terminus, which comprises two thymidine
nucleotide overhangs.

[0650] FIG. 9 compares a siNA construct synthesized by the method of the
invention described in Example 1, wherein an inverted deoxyabasic
succinate linker was used to generate a siNA having a 3'-inverted
deoxyabasic cap on the antisense strand of the siNA. This construct shows
improved activity compared to the control siNA construct consisting of
all ribonucleotides at every position except the 3'-terminus which
comprises two thymidine nucleotide overhangs.

[0651] FIG. 10 shows the results of an RNAi activity screen of chemically
modified siNA constructs including 3'-glyceryl modified siNA constructs
compared to an all RNA control siNA construct using a luciferase reporter
system. These chemically modified siNAs were compared in the luciferase
assay described herein at 1 nM and 10 nM concentration using an all RNA
siNA control (siGL2) having 3'-terminal dithymidine (TT) and its
corresponding inverted control (Inv siGL2). The background level of
luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.
As shown in the Figure, the 3'-terminal modified siNA constructs retain
significant RNAi activity compared to the unmodified control siNA (siGL2)
construct.

[0652] FIG. 11 shows the results of an RNAi activity screen of chemically
modified siNA constructs. The screen compared various combinations of
sense strand chemical modifications and antisense strand chemical
modifications. These chemically modified siNAs were compared in the
luciferase assay described herein at 1 nM and 10 nM concentration using
an all RNA siNA control (siGL2) having 3'-terminal dithymidine (TT) and
its corresponding inverted control (Inv siGL2). The background level of
luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.
As shown in the figure, the chemically modified Sirna/RPI 30063/30430,
Sirna/RPI 30433/30430, and Sirna/RPI 30063/30224 constructs retain
significant RNAi activity compared to the unmodified control siNA
construct. It should be noted that Sirna/RPI 30433/30430 is a siNA
construct having no ribonucleotides which retains significant RNAi
activity compared to the unmodified control siGL2 construct in vitro,
therefore, this construct is expected to have both similar RNAi activity
and improved stability in vivo compared to siNA constructs having
ribonucleotides.

[0653] FIG. 12 shows the results of an RNAi activity screen of chemically
modified siNA constructs. The screen compared various combinations of
sense strand chemical modifications and antisense strand chemical
modifications. These chemically modified siNAs were compared in the
luciferase assay described herein at 1 nM and 10 nM concentration using
an all RNA siNA control (siGL2) having 3'-terminal dithymidine (TT) and
its corresponding inverted control (Inv siGL2). The background level of
luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.
As shown in the figure, the chemically modified Sirna/RPI 30063/30224 and
Sirna/RPI 30063/30430 constructs retain significant RNAi activity
compared to the control siNA (siGL2) construct. In addition, the
antisense strand alone (Sirna/RPI 30430) and an inverted control
(Sirna/RPI 30227/30229), having matched chemistry to Sirna/RPI
(30063/30224) were compared to the siNA duplexes described above. The
antisense strand (Sirna/RPI 30430) alone provides far less inhibition
compared to the siNA duplexes using this sequence.

[0654] FIG. 13 shows the results of an RNAi activity screen of chemically
modified siNA constructs. The screen compared various combinations of
sense strand chemical modifications and antisense strand chemical
modifications. These chemically modified siNAs were compared in the
luciferase assay described herein at 1 nM and 10 nM concentration using
an all RNA siNA control (siGL2) having 3'-terminal dithymidine (TT) and
its corresponding inverted control (Inv siGL2). The background level of
luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.
In addition, an inverted control (Sirna/RPI 30226/30229, having matched
chemistry to Sirna/RPI 30222/30224) was compared to the siNA duplexes
described above. As shown in the figure, the chemically modified
Sirna/RPI 28251/30430, Sirna/RPI 28251/30224, and Sirna/RPI 30222/30224
constructs retain significant RNAi activity compared to the control siNA
construct, and the chemically modified Sirna/RPI 28251/30430 construct
demonstrates improved activity compared to the control siNA (siGL2)
construct.

[0655] FIG. 14 shows the results of an RNAi activity screen of chemically
modified siNA constructs including various 3'-terminal modified siNA
constructs compared to an all RNA control siNA construct using a
luciferase reporter system. These chemically modified siNAs were compared
in the luciferase assay described herein at 1 nM and 10 nM concentration
using an all RNA siNA control (siGL2) having 3'-terminal dithymidine (TT)
and its corresponding inverted control (Inv siGL2). The background level
of luciferase expression in the HeLa cells is designated by the "cells"
column. Sense and antisense strands of chemically modified siNA
constructs are shown by Sirna/RPI number (sense strand/antisense strand).
Sequences corresponding to these Sirna/RPI numbers are shown in Table I.
As shown in the figure, the chemically modified Sirna/RPI 30222/30546,
30222/30224, 30222/30551, 30222/30557 and 30222/30558 constructs retain
significant RNAi activity compared to the control siNA construct.

[0656]FIG. 15 shows the results of an RNAi activity screen of chemically
modified siNA constructs. The screen compared various combinations of
sense strand chemistries compared to a fixed antisense strand chemistry.
These chemically modified siNAs were compared in the luciferase assay
described herein at 1 nM and 10 nM concentration using an all RNA siNA
control (siGL2) having 3'-terminal dithymidine (TT) and its corresponding
inverted control (Inv siGL2). The background level of luciferase
expression in the HeLa cells is designated by the "cells" column. Sense
and antisense strands of chemically modified siNA constructs are shown by
Sirna/RPI number (sense strand/antisense strand). Sequences corresponding
to these Sirna/RPI numbers are shown in Table I. As shown in the figure,
the chemically modified Sirna/RPI 30063/30430, 30434/30430, and
30435/30430 constructs all demonstrate greater activity compared to the
control siNA (siGL2) construct.

Example 6

RNAi Activity Titration

[0657] A titration assay was performed to determine the lower range of
siNA concentration required for RNAi activity both in a control siNA
construct consisting of all RNA nucleotides containing two thymidine
nucleotide overhangs and a chemically modified siNA construct comprising
five phosphorothioate internucleotide linkages in both the sense and
antisense strands. The assay was performed as described above, however,
the siNA constructs were diluted to final concentrations between 2.5 nM
and 0.025 nM. Results are shown in FIG. 16. As shown in FIG. 16, the
chemically modified siNA construct shows a very similar concentration
dependent RNAi activity profile to the control siNA construct when
compared to an inverted siNA sequence control.

Example 7

siNA Design

[0658] siNA target sites were chosen by analyzing sequences of the target
RNA and optionally prioritizing the target sites on the basis of folding
(structure of any given sequence analyzed to determine siNA accessibility
to the target), by using a library of siNA molecules as described in
Example 4, or alternately by using an in vitro siNA system as described
in Example 9 herein. siNA molecules were designed that could bind each
target and are optionally individually analyzed by computer folding to
assess whether the siNA molecule can interact with the target sequence.
Varying the length of the siNA molecules can be chosen to optimize
activity. Generally, a sufficient number of complementary nucleotide
bases are chosen to bind to, or otherwise interact with, the target RNA,
but the degree of complementarity can be modulated to accommodate siNA
duplexes or varying length or base composition. By using such
methodologies, siNA molecules can be designed to target sites within any
known RNA sequence, for example those RNA sequences corresponding to the
any gene transcript.

[0659] Chemically modified siNA constructs are designed to provide
nuclease stability for systemic administration in vivo and/or improved
pharmacokinetic, localization, and delivery properties while preserving
the ability to mediate RNAi activity. Chemical modifications as described
herein are introduced synthetically using synthetic methods described
herein and those generally known in the art. The synthetic siNA
constructs are then assayed for nuclease stability in serum and/or
cellular/tissue extracts (e.g. liver extracts). The synthetic siNA
constructs are also tested in parallel for RNAi activity using an
appropriate assay, such as a luciferase reporter assay as described
herein or another suitable assay that can quantity RNAi activity.
Synthetic siNA constructs that possess both nuclease stability and RNAi
activity can be further modified and re-evaluated in stability and
activity assays. The chemical modifications of the stabilized active siNA
constructs can then be applied to any siNA sequence targeting any chosen
RNA and used, for example, in target screening assays to pick lead siNA
compounds for therapeutic development (see for example FIG. 27).

Example 8

Chemical Synthesis and Purification of siNA

[0660] siNA molecules can be designed to interact with various sites in
the RNA message, for example, target sequences within the RNA sequences
described herein. The sequence of one strand of the siNA molecule(s) is
complementary to the target site sequences described above. The siNA
molecules can be chemically synthesized using methods described herein.
Inactive siNA molecules that are used as control sequences can be
synthesized by scrambling the sequence of the siNA molecules such that it
is not complementary to the target sequence. Generally, siNA constructs
can by synthesized using solid phase oligonucleotide synthesis methods as
described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683;
5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117;
6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400;
6,111,086 all incorporated by reference herein in their entirety).

[0661] In a non-limiting example, RNA oligonucleotides are synthesized in
a stepwise fashion using the phosphoramidite chemistry as is known in the
art. Standard phosphoramidite chemistry involves the use of nucleosides
comprising any of 5'-O-dimethoxytrityl, 2'-O-tert-butyldimethylsilyl,
3'-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclic
amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,
and N2-isobutyryl guanosine). Alternately, 2'-O--Silyl Ethers can be used
in conjunction with acid-labile 2'-O-orthoester protecting groups in the
synthesis of RNA as described by Scaringe supra. Differing 2' chemistries
can require different protecting groups, for example 2'-deoxy-2'-amino
nucleosides can utilize N-phthaloyl protection as described by Usman et
al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its
entirety).

[0662] During solid phase synthesis, each nucleotide is added sequentially
(3'- to 5'-direction) to the solid support-bound oligonucleotide. The
first nucleoside at the 3'-end of the chain is covalently attached to a
solid support (e.g., controlled pore glass or polystyrene) using various
linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and
activator are combined resulting in the coupling of the second nucleoside
phosphoramidite onto the 5'-end of the first nucleoside. The support is
then washed and any unreacted 5'-hydroxyl groups are capped with a
capping reagent such as acetic anhydride to yield inactive 5'-acetyl
moieties. The trivalent phosphorus linkage is then oxidized to a more
stable phosphate linkage. At the end of the nucleotide addition cycle,
the 5'-O-protecting group is cleaved under suitable conditions (e.g.,
acidic conditions for trityl-based groups and Fluoride for silyl-based
groups). The cycle is repeated for each subsequent nucleotide.

[0663] Modification of synthesis conditions can be used to optimize
coupling efficiency, for example by using differing coupling times,
differing reagent/phosphoramidite concentrations, differing contact
times, differing solid supports and solid support linker chemistries
depending on the particular chemical composition of the siNA to be
synthesized. Deprotection and purification of the siNA can be performed
as is generally described in Deprotection and purification of the siNA
can be performed as is generally described in Usman et al., U.S. Pat. No.
5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon
et al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No.
6,303,773, or Scaringe supra, incorporated by reference herein in their
entireties. Additionally, deprotection conditions can be modified to
provide the best possible yield and purity of siNA constructs. For
example, applicant has observed that oligonucleotides comprising
2'-deoxy-2'-fluoro nucleotides can degrade under inappropriate
deprotection conditions. Such oligonucleotides are deprotected using
aqueous methylamine at about 35° C. for 30 minutes. If the
2'-deoxy-2'-fluoro containing oligonucleotide also comprises
ribonucleotides, after deprotection with aqueous methylamine at about
35° C. for 30 minutes, TEA-HF is added and the reaction maintained
at about 65° C. for an additional 15 minutes.

Example 9

RNAi In Vitro Assay to Assess siNA Activity

[0664] An in vitro assay that recapitulates RNAi in a cell free system is
used to evaluate siNA constructs specific to target RNA. The assay
comprises the system described by Tuschl et al., 1999, Genes and
Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33
adapted for use with target RNA. A Drosophila extract derived from
syncytial blastoderm is used to reconstitute RNAi activity in vitro.
Target RNA is generated via in vitro transcription from an appropriate
plasmid using T7 RNA polymerase or via chemical synthesis as described
herein. Sense and antisense siNA strands (for example 20 uM each) are
annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM
HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C.
followed by 1 hour at 37° C., then diluted in lysis buffer (for
example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM
magnesium acetate). Annealing can be monitored by gel electrophoresis on
an agarose gel in TBE buffer and stained with ethidium bromide. The
Drosophila lysate is prepared using zero to two-hour-old embryos from
Oregon R flies collected on yeasted molasses agar that are dechorionated
and lysed. The lysate is centrifuged and the supernatant isolated. The
assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA
(10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing
siNA (10 nM final concentration). The reaction mixture also contains 10
mM creatine phosphate, 10 ug.ml creatine phosphokinase, 100 um GTP, 100
uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and
100 uM of each amino acid. The final concentration of potassium acetate
is adjusted to 100 mM. The reactions are pre-assembled on ice and
preincubated at 25° C. for 10 minutes before adding RNA, then
incubated at 25° C. for an additional 60 minutes. Reactions are
quenched with 4 volumes of 1.25× Passive Lysis Buffer (Promega).
Target RNA cleavage is assayed by RT-PCR analysis or other methods known
in the art and are compared to control reactions in which siNA is omitted
from the reaction.

[0665] Alternately, internally-labeled target RNA for the assay is
prepared by in vitro transcription in the presence of [alpha-32P]
CTP, passed over a G 50 Sephadex column by spin chromatography and used
as target RNA without further purification. Optionally, target RNA is
5'-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are
performed as described above and target RNA and the specific RNA cleavage
products generated by RNAi are visualized on an autoradiograph of a gel.
The percentage of cleavage is determined by Phosphor Imager®
quantitation of bands representing intact control RNA or RNA from control
reactions without siNA and the cleavage products generated by the assay.

[0666] In one embodiment, this assay is used to determine target sites the
RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA
constructs are screened for RNAi mediated cleavage of the RNA target, for
example, by analyzing the assay reaction by electrophoresis of labeled
target RNA, or by northern blotting, as well as by other methodology well
known in the art.

Example 10

Nucleic Acid Inhibition of Target RNA In Vivo

[0667] siNA molecules targeted to the target RNA are designed and
synthesized as described above. These nucleic acid molecules can be
tested for cleavage activity in vivo, for example, using the following
procedure.

[0668] Two formats are used to test the efficacy of siNAs targeting a
particular gene transcipt. First, the reagents are tested on target
expressing cells (e.g., HeLa), to determine the extent of RNA and protein
inhibition. siNA reagents are selected against the RNA target. RNA
inhibition is measured after delivery of these reagents by a suitable
transfection agent to cells. Relative amounts of target RNA are measured
versus actin using real-time PCR monitoring of amplification (eg., ABI
7700 Taqman®). A comparison is made to a mixture of oligonucleotide
sequences made to unrelated targets or to a randomized siNA control with
the same overall length and chemistry, but with randomly substituted
nucleotides at each position. Primary and secondary lead reagents are
chosen for the target and optimization performed. After an optimal
transfection agent concentration is chosen, a RNA time-course of
inhibition is performed with the lead siNA molecule. In addition, a
cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

[0669] Cells (e.g., HeLa) are seeded, for example, at 1×105
cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before
transfection. siNA (final concentration, for example 20 nM) and cationic
lipid (e.g., final concentration 2 μg/ml) are complexed in EGM basal
media (Biowhittaker) at 37° C. for 30 mins in polystyrene tubes.
Following vortexing, the complexed siNA is added to each well and
incubated for the times indicated. For initial optimization experiments,
cells are seeded, for example, at 1×103 in 96 well plates and
siNA complex added as described. Efficiency of delivery of siNA to cells
is determined using a fluorescent siNA complexed with lipid. Cells in
6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and
fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake
of siNA is visualized using a fluorescent microscope.

Taqman and Lightcycler Quantification of mRNA

[0670] Total RNA is prepared from cells following siNA delivery, for
example, using Qiagen RNA purification kits for 6-well or Rneasy
extraction kits for 96-well assays. For Taqman analysis, dual-labeled
probes are synthesized with the reporter dye, FAM or JOE, covalently
linked at the 5'-end and the quencher dye TAMRA conjugated to the 3'-end.
One-step RT-PCR amplifications are performed on, for example, an ABI
PRISM 7700 Sequence Detector using 50 μA reactions consisting of 10
μA total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM
probe, 1× TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5
mM MgCl2, 300 μM each dATP, dCTP, dGTP, and dTTP, 10 U RNase
Inhibitor (Promega), 1.25 U AmpliTaq Gold (PE-Applied Biosystems) and 10
U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions
can consist of 30 min at 48° C., 10 min at 95° C., followed
by 40 cycles of 15 sec at 95° C. and 1 min at 60° C.
Quantitation of mRNA levels is determined relative to standards generated
from serially diluted total cellular RNA (300, 100, 33, 11 ng/r×n)
and normalizing to β-actin or GAPDH mRNA in parallel TaqMan
reactions. For each gene of interest an upper and lower primer and a
fluorescently labeled probe are designed. Real time incorporation of SYBR
Green I dye into a specific PCR product can be measured in glass
capillary tubes using a lightcyler. A standard curve is generated for
each primer pair using control cRNA. Values are represented as relative
expression to GAPDH in each sample.

Western Blotting

[0671] Nuclear extracts can be prepared using a standard micro preparation
technique (see for example Andrews and Faller, 1991, Nucleic Acids
Research, 19, 2499). Protein extracts from supernatants are prepared, for
example using TCA precipitation. An equal volume of 20% TCA is added to
the cell supernatant, incubated on ice for 1 hour and pelleted by
centrifugation for 5 minutes. Pellets are washed in acetone, dried and
resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris
NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts)
polyacrylamide gel and transferred onto nitro-cellulose membranes.
Non-specific binding can be blocked by incubation, for example, with 5%
non-fat milk for 1 hour followed by primary antibody for 16 hour at
4° C. Following washes, the secondary antibody is applied, for
example (1:10,000 dilution) for 1 hour at room temperature and the signal
detected with SuperSignal reagent (Pierce).

Example 11

Animal Models

[0672] Various animal models can be used to screen siNA constructs in vivo
as are known in the art, for example those animal models that are used to
evaluate other nucleic acid technologies such as enzymatic nucleic acid
molecules (ribozymes) and/or antisense. Such animal models are used to
test the efficacy of siNA molecules described herein. In a non-limiting
example, siNA molecules that are designed as anti-angiogenic agents can
be screened using animal models. There are several animal models
available in which to test the anti-angiogenesis effect of nucleic acids
of the present invention, such as siNA, directed against genes associated
with angiogenesis and/or metastais, such as VEGFR (e.g., VEGFR1, VEGFR2,
and VEGFR3) genes. Typically a corneal model has been used to study
angiogenesis in rat and rabbit, since recruitment of vessels can easily
be followed in this normally avascular tissue (Pandey et al., 1995
Science 268: 567-569). In these models, a small Teflon or Hydron disk
pretreated with an angiogenesis factor (e.g. bFGF or VEGF) is inserted
into a pocket surgically created in the cornea. Angiogenesis is monitored
3 to 5 days later. siNA molecules directed against VEGFR mRNAs would be
delivered in the disk as well, or dropwise to the eye over the time
course of the experiment. In another eye model, hypoxia has been shown to
cause both increased expression of VEGF and neovascularization in the
retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92: 905-909;
Shweiki et al., 1992 J. Clin. Invest. 91: 2235-2243).

[0674] The cornea model, described in Pandey et al. supra, is the most
common and well characterized anti-angiogenic agent efficacy screening
model. This model involves an avascular tissue into which vessels are
recruited by a stimulating agent (growth factor, thermal or alkalai burn,
endotoxin). The corneal model utilizes the intrastromal corneal
implantation of a Teflon pellet soaked in a VEGF-Hydron solution to
recruit blood vessels toward the pellet, which can be quantitated using
standard microscopic and image analysis techniques. To evaluate their
anti-angiogenic efficacy, siNA molecules are applied topically to the eye
or bound within Hydron on the Teflon pellet itself. This avascular cornea
as well as the Matrigel model (described below) provide for low
background assays. While the corneal model has been performed extensively
in the rabbit, studies in the rat have also been conducted.

[0675] The mouse model (Passaniti et al., supra) is a non-tissue model
which utilizes Matrigel, an extract of basement membrane (Kleinman et
al., 1986) or Millipore® filter disk, which can be impregnated with
growth factors and anti-angiogenic agents in a liquid form prior to
injection. Upon subcutaneous administration at body temperature, the
Matrigel or Millipore® filter disk forms a solid implant. VEGF
embedded in the Matrigel or Millipore® filter disk is used to recruit
vessels within the matrix of the Matrigel or Millipore® filter disk
which can be processed histologically for endothelial cell specific vWF
(factor VIII antigen) immunohistochemistry, Trichrome-Masson stain, or
hemoglobin content. Like the cornea, the Matrigel or Millipore®
filter disk are avascular; however, it is not tissue. In the Matrigel or
Millipore® filter disk model, siNA molecules are administered within
the matrix of the Matrigel or Millipore® filter disk to test their
anti-angiogenic efficacy. Thus, delivery issues in this model, as with
delivery of siNA molecules by Hydron-coated Teflon pellets in the rat
cornea model, may be less problematic due to the homogeneous presence of
the siNA within the respective matrix.

[0676] The Lewis lung carcinoma and B-16 murine melanoma models are well
accepted models of primary and metastatic cancer and are used for initial
screening of anti-cancer agents. These murine models are not dependent
upon the use of immunodeficient mice, are relatively inexpensive, and
minimize housing concerns. Both the Lewis lung and B-16 melanoma models
involve subcutaneous implantation of approximately 106 tumor cells
from metastatically aggressive tumor cell lines (Lewis lung lines 3LL or
D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice. Alternatively, the
Lewis lung model can be produced by the surgical implantation of tumor
spheres (approximately 0.8 mm in diameter). Metastasis also may be
modeled by injecting the tumor cells directly intraveneously. In the
Lewis lung model, microscopic metastases can be observed approximately 14
days following implantation with quantifiable macroscopic metastatic
tumors developing within 21-25 days. The B-16 melanoma exhibits a similar
time course with tumor neovascularization beginning 4 days following
implantation. Since both primary and metastatic tumors exist in these
models after 21-25 days in the same animal, multiple measurements can be
taken as indices of efficacy. Primary tumor volume and growth latency as
well as the number of micro- and macroscopic metastatic lung foci or
number of animals exhibiting metastases can be quantitated. The percent
increase in lifespan can also be measured. Thus, these models would
provide suitable primary efficacy assays for screening systemically
administered siNA molecules and siNA formulations.

[0677] In the Lewis lung and B-16 melanoma models, systemic
pharmacotherapy with a wide variety of agents usually begins 1-7 days
following tumor implantation/inoculation with either continuous or
multiple administration regimens. Concurrent pharmacokinetic studies can
be performed to determine whether sufficient tissue levels of siNA can be
achieved for pharmacodynamic effect to be expected. Furthermore, primary
tumors and secondary lung metastases can be removed and subjected to a
variety of in vitro studies (i.e. target RNA reduction).

[0678] Ohno-Matsui et al., 2002, Am. J. Pathology, 160, 711-719 describe a
model of severe proliferative retinopathy and retinal detachment in mice
under inducible expression of vascular endothelial growth factor. In this
model, expression of a VEGF transgene results in elevated levels of
ocular VEGF that is associated with severe proliferative retinopathy and
retinal detachment. Furthermore, Mori et al., 2001, J. Cellular
Physiology, 188, 253-263, describe a model of laser induced choroidal
neovascularization that can be used in conjunction with intravitreous or
subretianl injection of siNA molecules of the invention to evaluate the
efficacy of siNA treatment of severe proliferative retinopathy and
retinal detachment.

[0680] The purpose of this study was to assess the anti-angiogenic
activity of siNA targeted against VEGFR1, using the rat cornea model of
VEGF induced angiogenesis discussed in Example 11 above). The siNA
molecules shown in FIG. 23 have matched inverted controls which are
inactive since they are not able to interact with the RNA target. The
siNA molecules and VEGF were co-delivered using the filter disk method.
Nitrocellulose filter disks (Millipore®) of 0.057 diameter were
immersed in appropriate solutions and were surgically implanted in rat
cornea as described by Pandey et al., supra.

[0681] The stimulus for angiogenesis in this study was the treatment of
the filter disk with 30 μM VEGF which is implanted within the cornea's
stroma. This dose yields reproducible neovascularization stemming from
the pericorneal vascular plexus growing toward the disk in a
dose-response study 5 days following implant. Filter disks treated only
with the vehicle for VEGF show no angiogenic response. The siNA were
co-adminstered with VEGF on a disk in three different siNA
concentrations. One concern with the simultaneous administration is that
the siNA would not be able to inhibit angiogenesis since VEGF receptors
can be stimulated. However, Applicant has observed that in low VEGF
doses, the neovascular response reverts to normal suggesting that the
VEGF stimulus is essential for maintaining the angiogenic response.
Blocking the production of VEGF receptors using simultaneous
administration of anti-VEGF-R mRNA siNA could attenuate the normal
neovascularization induced by the filter disk treated with VEGF.

[0688] Animals are housed in groups of two. Feed, water, temperature and
humidity are determined according to Pharmacology Testing Facility
performance standards (SOP's) which are in accordance with the 1996 Guide
for the Care and Use of Laboratory Animals (NRC). Animals are acclimated
to the facility for at least 7 days prior to experimentation. During this
time, animals are observed for overall health and sentinels are bled for
baseline serology.

Experimental Groups

[0689] Each solution (VEGF and siNAs) was prepared as a 1× solution
for final concentrations shown in the experimental groups described in
Table III.

siNA Annealing Conditions

[0690] siNA sense and antisense strands are annealed for 1 minute in
H2O at 1.67 mg/mL/strand followed by a 1 hour incubation at
37° C. producing 3.34 mg/mL of duplexed siNA. For the 20 μg/eye
treatment, 6 μLs of the 3.34 mg/mL duplex is injected into the eye
(see below). The 3.34 mg/mL duplex siNA can then be serially diluted for
dose response assays.

[0692] The rat corneal model used in this study was a modified from Koch
et al. Supra and Pandey et al., supra. Briefly, corneas were irrigated
with 0.5% povidone iodine solution followed by normal saline and two
drops of 2% lidocaine. Under a dissecting microscope (Leica MZ-6), a
stromal pocket was created and a presoaked filter disk (see above) was
inserted into the pocket such that its edge was 1 mm from the corneal
limbus.

Intraconjunctival Injection of Test Solutions

[0693] Immediately after disk insertion, the tip of a 40-50 μm OD
injector (constructed in our laboratory) was inserted within the
conjunctival tissue 1 mm away from the edge of the corneal limbus that
was directly adjacent to the VEGF-soaked filter disk. Six hundred
nanoliters of test solution (siNA, inverted control or sterile water
vehicle) were dispensed at a rate of 1.2 μL/min using a syringe pump
(Kd Scientific). The injector was then removed, serially rinsed in 70%
ethanol and sterile water and immersed in sterile water between each
injection. Once the test solution was injected, closure of the eyelid was
maintained using microaneurism clips until the animal began to recover
gross motor activity. Following treatment, animals were warmed on a
heating pad at 37° C.

Quantitation of Angiogenic Response

[0694] Five days after disk implantation, animals were euthanized
following administration of 0.4 mg/kg atropine and corneas were digitally
imaged. The neovascular surface area (NSA, expressed in pixels) was
measured postmortem from blood-filled corneal vessels using computerized
morphometry (Image Pro Plus, Media Cybernetics, v2.0). The individual
mean NSA was determined in triplicate from three regions of identical
size in the area of maximal neovascularization between the filter disk
and the limbus. The number of pixels corresponding to the blood-filled
corneal vessels in these regions was summated to produce an index of NSA.
A group mean NSA was then calculated. Data from each treatment group were
normalized to VEGF/siNA vehicle-treated control NSA and finally expressed
as percent inhibition of VEGF-induced angiogenesis.

Statistics

[0695] After determining the normality of treatment group means, group
mean percent inhibition of VEGF-induced angiogenesis was subjected to a
one-way analysis of variance. This was followed by two post-hoc tests for
significance including Dunnett's (comparison to VEGF control) and
Tukey-Kramer (all other group mean comparisons) at alpha=0.05.
Statistical analyses were performed using JMP v.3.1.6 (SAS Institute).

[0696] Results of the study are graphically represented in FIGS. 23 and
76. As shown in FIG. 23, VEGFr1 site 4229 active siNA (Sirna/RPI
29695/29699) at three concentrations were effective at inhibiting
angiogenesis compared to the inverted siNA control (Sirna/RPI
29983/29984) and the VEGF control. A chemically modified version of the
VEGFr1 site 4229 active siNA comprising a sense strand having
2'-deoxy-2'-fluoro pyrimidines and ribo purines with 5' and 3' terminal
inverted deoxyabasic residues and an antisense strand having
2'-deoxy-2'-fluoro pyrimidines and ribo purines with a terminal
3'-phosphorothioate internucleotide linkage (Sirna/RPI 30196/30416),
showed similar inhibition. Furthermore, VEGFrl site 349 active siNA
having "Stab 9/10" chemistry (Sirna #31270/31273) was tested for
inhibition of VEGF-induced angiogenesis at three different concentrations
(2.0 ug, 1.0 ug, and 0.1 μg dose response) as compared to a matched
chemistry inverted control siNA construct (Sirna #31276/31279) at each
concentration and a VEGF control in which no siNA was administered. As
shown in FIG. 76, the active siNA construct having "Stab 9/10" chemistry
(Sirna #31270/31273) is highly effective in inhibiting VEGF-induced
angiogenesis in the rat corneal model compared to the matched chemistry
inverted control siNA at concentrations from 0.1 μg to 2.0 ug. These
results demonstrate that siNA molecules having different chemically
modified compositions, such as the modifications described herein, are
capable of significantly inhibiting angiogenesis in vivo.

Example 13

Inhibition of HBV Using siNA Molecules of the Invention

[0697] Transfection of HepG2 Cells with psHBV-1 and siNA

[0698] The human hepatocellular carcinoma cell line Hep G2 was grown in
Dulbecco's modified Eagle media supplemented with 10% fetal calf serum, 2
mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 25
mM Hepes, 100 units penicillin, and 100 μg/ml streptomycin. To
generate a replication competent cDNA, prior to transfection the HBV
genomic sequences are excised from the bacterial plasmid sequence
contained in the psHBV-1 vector. Other methods known in the art can be
used to generate a replication competent cDNA. This was done with an
EcoRI and Hind III restriction digest. Following completion of the
digest, a ligation was performed under dilute conditions (20 μg/ml) to
favor intermolecular ligation. The total ligation mixture was then
concentrated using Qiagen spin columns.

siNA Activity Screen and Dose Response Assay

[0699] Transfection of the human hepatocellular carcinoma cell line, Hep
G2, with replication-competent HBV DNA results in the expression of HBV
proteins and the production of virions. To test the efficacy of siNAs
targeted against HBV RNA, several siNA duplexes targeting different sites
within HBV pregenomic RNA were co-transfected with HBV genomic DNA once
at 25 nM with lipid at 12.5 ug/ml into Hep G2 cells, and the subsequent
levels of secreted HBV surface antigen (HBsAg) were analyzed by ELISA
(see FIG. 24). Inverted sequence duplexes were used as negative controls.
Subsequently, dose response studies were performed in which the siNA
duplexes were co-transfected with HBV genomic DNA at 0.5, 5, 10 and 25 nM
with lipid at 12.5 ug/ml into Hep G2 cells, and the subsequent levels of
secreted HBV surface antigen (HBsAg) were analyzed by ELISA (see FIG.
25).

Analysis of HBsAg Levels Following siNA Treatment

[0700] To determine siNA activity, HbsAg levels were measured following
transfection with siNA. Immulon 4 (Dynax) microtiter wells were coated
overnight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay)
at 1 μg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5).
The wells were then washed 4× with PBST (PBS, 0.05% Tween® 20)
and blocked for 1 hr at 37° C. with PBST, 1% BSA. Following
washing as above, the wells were dried at 37° C. for 30 min.
Biotinylated goat ant-HBsAg (Accurate YVS1807) was diluted 1:1000 in PBST
and incubated in the wells for 1 hr. at 37° C. The wells were
washed 4× with PBST. Streptavidin/Alkaline Phosphatase Conjugate
(Pierce 21324) was diluted to 250 ng/ml in PBST, and incubated in the
wells for 1 hr. at 37° C. After washing as above, p-nitrophenyl
phosphate substrate (Pierce 37620) was added to the wells, which were
then incubated for 1 hour at 37° C. The optical density at 405 nm
was then determined. Results of the HBV screen study are summarized in
FIG. 24, whereas the results of a dose response assay using lead siNA
constructs targeting sites 262 and 1580 of the HBV pregenomic RNA are
shown in FIG. 25. As shown in FIG. 25, the siNA constructs targeting
sites 262 and 1580 of HBV RNA provides significant dose response
inhibition of viral replication/activity when compared to inverted siNA
controls.

[0702] Four different siNA constructs having different stabilization
chemistries were compared to an unstabilized siRNA construct in a dose
response time course HBsAg assay, the results of which are shown in FIGS.
28-31. The different constructs were compared to an unstabilized
ribonucleotide control siRNA construct (Sirna/RPI#30287/30298) at
different concentrations (5 nM, 10 nM, 25 nM, 50 nM, and 100 nM) over the
course of nine days. Activity based on HBsAg levels was determined at day
3, day 6, and day 9. The "Stab 4/5" (Table IV) constructs comprise a
sense strand (Sirna/RPI#30355) having 2'-deoxy-2'-fluoro pyrimidine
nucleotides and purine ribonucleotides with 5' and 3' terminal inverted
deoxyabasic residues and an antisense strand (Sirna/RPI#30366) having
2'-deoxy-2'-fluoro pyrimidine nucleotides and purine ribonucleotides with
a terminal 3' phosphorothioate linkage (data shown in FIG. 28). The
"Stab7/8" (Table IV) constructs comprise a sense strand (Sirna/RPI#30612)
having 2'-deoxy-2'-fluoro pyrimidine nucleotides and 2'-deoxy purine
nucleotides with 5' and 3' terminal inverted deoxyabasic residues and an
antisense strand (Sirna/RPI#30620) having 2'-deoxy-2'-fluoro pyrimidine
nucleotides and 2'-O-methyl purine nucleotides with a terminal 3'
phosphorothioate linkage (data shown in FIG. 29). The "Stab7/11 (Table
IV) constructs comprise a sense (Sirna/RPI#30612) strand having
2'-deoxy-2'-fluoro pyrimidine nucleotides and 2'-deoxy purine nucleotides
with 5' and 3' terminal inverted deoxyabasic residues and an antisense
strand (Sirna/RPI#31175) having 2'-deoxy-2'-fluoro pyrimidine nucleotides
and 2'-deoxy purine nucleotides with a terminal 3' phosphorothioate
linkage (data shown in FIG. 30). The "Stab9/10 (Table IV) constructs
comprise a sense (Sirna/RPI#31335) strand having ribonucleotides with 5'
and 3' terminal inverted deoxyabasic residues and an antisense strand
(Sirna/RPI#31337) having ribonucleotides with a terminal 3'
phosphorothioate linkage (data shown in FIG. 31). As shown in FIGS.
28-31, the chemically stabilized siNA constructs all show significantly
greater inhibition of HBV antigen in a dose dependent manner over the
time course experiment compared to the unstabilized siRNA construct.

[0703] A second study was performed using the stab 4/5 (Sirna
30355/30366), stab 7/8 (Sirna 30612/30620), and stab 7/11 (Sirna
30612/31175) siNA constructs described above to examine the duration of
effect of the modified siNA constructs out to 21 days post transfection
compared to an all RNA control siNA (Sirna 30287/30298). A single
transfection was performed with siRNAs targeted to HBV site 1580 and the
culture media was subsequently replaced every three days. Secreted HBsAg
levels were monitored for at 3, 6, 9, 12, 15, 18 and 21 days
post-transfection. FIG. 77 shows activity of siNAs in reduction of HBsAg
levels compared to matched inverted controls at A. 3 days, B. 9 days, and
C. 21 days post transfection. Also shown is the corresponding percent
inhibition as function of time at siNA concentrations of D. 100 nM, E. 50
nM, and F. 25 nM.

Example 14

Inhibition of HCV Using siNA Molecules of the Invention

[0704] siNA Inhibition of a Chimeric HCV/Poliovirus in HeLa Cells

[0705] Inhibition of a chimeric HCV/Poliovirus was investigated using 21
nucleotide siNA duplexes in HeLa cells. Seven siNA constructs were
designed that target three regions in the highly conserved 5'
untranslated region (UTR) of HCV RNA. The siNAs were screened in two cell
culture systems dependent upon the 5'-UTR of HCV; one requires
translation of an HCV/luciferase gene, while the other involves
replication of a chimeric HCV/poliovirus (PV) (see Blatt et al., U.S.
Ser. No. 09/740,332, filed Dec. 18, 2000, incorporated by reference
herein). Two siNAs (29579/29586; 29578/29585) targeting the same region
(shifted by one nucleotide) are active in both systems (see FIG. 32) as
compared with inverse control siNA (29593/29600). For example, a >85%
reduction in HCVPV replication was observed in siNA-treated cells
compared to an inverse siNA control (FIG. 32) with an IC50=˜2.5 nM
(FIG. 33). To develop nuclease-resistant siNA for in vivo applications,
siNAs can be modified to contain stabilizing chemical modifications. Such
modifications include phosphorothioate linkages (P═S), 2'-O-methyl
nucleotides, 2'-fluoro (F) nucleotides, 2'-deoxy nucleotides, universal
base nucleotides, 5' and/or 3' end modifications and a variety of other
nucleotide and non-nucleotide modifications, in one or both siNA strands.
Several of these constructs were tested in the HCV/poliovirus chimera
system, demonstrating significant reduction in viral replication (FIGS.
34-37). siNA constructs shown in FIGS. 34-37 are referred to by
Sirna/RPI#s that are cross referenced to Table III, which shows the
sequence and chemical modifications of the constructs. siNA activity is
compared to relevant controls (untreated cells, scrambled/inactive
control sequences, or transfection controls). As shown in the Figures,
siNA constructs of the invention provide potent inhibition of HCV RNA in
the HCV/poliovirus chimera system. As such, siNA constructs, inlcuding
chemically modified, nuclease resistant siNA molecules, represent an
important class of therapeutic agents for treating chronic HCV infection.

siNA Inhibition of a HCV RNA Expression in a HCV Replicon System

[0706] In addition, a HCV replicon system was used to test the efficacy of
siNAs targeting HCV RNA. The reagents are tested in cell culture using
Huh7 cells (see for example Randall et al., 2003, PNAS USA, 100, 235-240)
to determine the extent of RNA and protein inhibition. siNA were selected
against the HCV target as described herein. RNA inhibition was measured
after delivery of these reagents by a suitable transfection agent to Huh7
cells. Relative amounts of target RNA are measured versus actin using
real-time PCR monitoring of amplification (eg., ABI 7700 Taqman®). A
comparison is made to a mixture of oligonucleotide sequences designed to
target unrelated targets or to a randomized siNA control with the same
overall length and chemistry, but with randomly substituted nucleotides
at each position. Primary and secondary lead reagents were chosen for the
target and optimization performed. After an optimal transfection agent
concentration is chosen, a RNA time-course of inhibition is performed
with the lead siNA molecule. In addition, a cell-plating format can be
used to determine RNA inhibition. A non-limiting example of a multiple
target screen to assay siNA mediated inhibition of HCV RNA is shown in
FIG. 38. siNA reagents (Table I) were transfected at 25 nM into Huh7
cells and HCV RNA quantitated compared to untreated cells ("cells" column
in the figure) and cells transfected with lipofectamine ("LFA2K" column
in the figure). As shown in the Figure, several siNA constructs show
significant inhibition of HCV RNA expression in the Huh7 replicon system.
Chemically modified siNA constructs were then screened as described
above, with a non-limiting example of a Stab 7/8 (see Table IV) chemistry
siNA construct screen shown in FIG. 40. A follow up dose response study
using chemically modified siNA constructs (Stab 4/5, see Table IV) at
concentrations of 5 nM, 10 nM, 25 nM and 100 nM compared to matched
chemistry inverted controls is shown in FIG. 39, whereas a dose response
study for Stab 7/8 constructs at concentrations of 5 nM, 10 nM, 25 nM, 50
nM and 100 nM compared to matched chemistry inverted controls is shown in
FIG. 41. A separate direct screen of Stab 7/8 constructs targeting HCV
RNA that identified stabilized siNA constructs with potent activity is
shown in FIG. 86.

Example 15

Target Discovery in Mammalian Cells Using siNA Molecules

[0707] In a non-limiting example, compositions and methods of the
invention are used to discover genes involved in a process of interest
within mammalian cells, such as cell growth, proliferation, apoptosis,
morphology, angiogenesis, differentiation, migration, viral
multiplication, drug resistance, signal transduction, cell cycle
regulation, or temperature sensitivity or other process. First, a
randomized siNA library is generated. These constructs are inserted into
a vector capable of expressing a siNA from the library inside mammalian
cells. Alternately, a pool of synthetic siNA molecules is generated.

Reporter System

[0708] In order to discover genes playing a role in the expression of
certain proteins, such as proteins involved in a cellular process
described herein, a readily assayable reporter system is constructed in
which a reporter molecule is co-expressed when a particular protein of
interest is expressed. The reporter system consists of a plasmid
construct bearing a gene coding for a reporter gene, such as Green
Fluorescent Protein (GFP) or other reporter proteins known and readily
available in the art. The promoter region of the GFP gene is replaced by
a portion of a promoter for the protein of interest sufficient to direct
efficient transcription of the GFP gene. The plasmid can also contain a
drug resistance gene, such as neomycin resistance, in order to select
cells containing the plasmid.

Host Cell Lines for Target Discovery

[0709] A cell line is selected as host for target discovery. The cell line
is preferably known to express the protein of interest, such that
upstream genes controlling the expression of the protein can be
identified when modulated by a siNA construct expressed therein. The
cells preferably retain protein expression characteristics in culture.
The reporter plasmid is transfected into cells, for example, using a
cationic lipid formulation. Following transfection, the cells are
subjected to limiting dilution cloning, for example, under selection by
600 μg/mL Geneticin. Cells retaining the plasmid survive the Geneticin
treatment and form colonies derived from single surviving cells. The
resulting clonal cell lines are screened by flow cytometry for the
capacity to upregulate GFP production. Treating the cells with, for
example, sterilized M9 bacterial medium in which Pseudomonas aeruginosa
had been cultured (Pseudomonas conditioned medium, PCM) is used to induce
the promoter. The PCM is supplemented with phorbol myristate acetate
(PMA). A clonal cell line highly responsive to promoter induction is
selected as the reporter line for subsequent studies.

siNA Library Construction

[0710] A siNA library was constructed with oligonucletides containing
hairpin siNA constructs having randomized antisense regions and self
complementary sense regions. The library is generated synthesizing siNA
constructs having randomized sequence. Alternately, the siNA libraries
are constructed as described in Usman et al., U.S. Ser. No. 60/402,996
(incorporated by reference herein) Oligo sequence 5' and 3' of the siNA
contains restriction endonuclease cleavage sites for cloning. The 3'
trailing sequence forms a stem-loop for priming DNA polymerase extension
to form a hairpin structure. The hairpin DNA construct is melted at
90° C. allowing DNA polymerase to generate a dsDNA construct. The
double-stranded siNA library is cloned into, for example, a U6+27
transcription unit located in the 5' LTR region of a retroviral vector
containing the human nerve growth factor receptor (hNGFr) reporter gene.
Positioning the U6+27/siNA transcription unit in the 5' LTR results in a
duplication of the transcription unit when the vector integrates into the
host cell genome. As a result, the siNA is transcribed by RNA polymerase
III from U6+27 and by RNA polymerase II activity directed by the 5' LTR.
The siNA library is packaged into retroviral particles that are used to
infect and transduce clonal cells selected above. Assays of the hNGFr
reporter are used to indicate the percentage of cells that incorporated
the siNA construct. By randomized region is meant a region of completely
random sequence and/or partially random sequence. By completely random
sequence is meant a sequence wherein theoretically there is equal
representation of A, T, G and C nucleotides or modified derivatives
thereof, at each position in the sequence. By partially random sequence
is meant a sequence wherein there is an unequal representation of A, T, G
and C nucleotides or modified derivatives thereof, at each position in
the sequence. A partially random sequence can therefore have one or more
positions of complete randomness and one or more positions with defined
nucleotides.

Enriching for Non-Responders to Induction

[0711] Sorting of siNA library-containing cells is performed to enrich for
cells that produce less reporter GFP after treatment with the promoter
inducers PCM and PMA. Lower GFP production cancan be due to RNAi activity
against genes involved in the activation of the mucin promoter.
Alternatively, siNA can directly target the mucin/GFP transcript
resulting in reduced GFP expression.

[0712] Cells are seeded at a certain density, such as 1×106 per
150 cm2 style cell culture flasks and grown in the appropriate cell
culture medium with fetal bovine serum. After 72 hours, the cell culture
medium is replaced with serum-free medium. After 24 hours of serum
deprivation, the cells are treated with serum-containing medium
supplemented with PCM (to 40%) and PMA (to 50 nM) to induced GFP
production. After 20 to 22 hours, cells are monitored for GFP level on,
for example, a FACStar Plus cell sorter. Sorting is performed if
≧90% of siNA library cells from an unsorted control sample were
induced to produce GFP above background levels. Two cell fractions are
collected in each round of sorting. Following the appropriate round of
sorting, the Ml fraction is selected to generate a database of siNA
molecules present in the sorted cells.

Recovery of siNA Sequence from Sorted Cells

[0713] Genomic DNA is obtained from sorted siNA library cells by standard
methods. Nested polymerase chain reaction (PCR) primers that hybridized
to the retroviral vector 5' and 3' of the siNA are used to recover and
amplify the siNA sequences from the particular clone of library cell DNA.
The PCR product is ligated into a bacterial cloning vector. The recovered
siNA library in plasmid form can be used to generate a database of siNA
sequences. For example, the library is cloned into E. coli. DNA is
prepared by plasmid isolation from bacterial colonies or by direct colony
PCR and siNA sequence is determined. A second method can use the siNA
library to transfect cloned cells. Clonal lines of stably transfected
cells are established and induced with, for example, PCM and PMA. Those
lines which fail to respond to GFP induction are probed by PCR for single
siNA integration events. The unique siNA sequences obtained by both
methods are added to a Target Sequence Tag (TST) database.

Bioinformatics

[0714] The antisense region sequences of the isolated siNA constructs are
compared to public and private gene data banks. Gene matches are compiled
according to perfect and imperfect matches. Potential gene targets are
categorized by the number of different siNA sequences matching each gene.
Genes with more than one perfect siNA match are selected for Target
Validation studies.

Validation of the Target Gene

[0715] To validate a target as a regulator of protein expression, siNA
reagents are designed to the target gene cDNA sequence from Genbank. The
siNA reagents are complexed with a cationic lipid formulation prior to
administration to cloned cells at appropriate concentrations (e.g. 5-50
nM or less). Cells are treated with siNA reagents, for example from 72 to
96 hours. Before the termination of siNA treatment, PCM (to 40%) and PMA
(to 50 nM), for example, are added to induce the promoter. After twenty
hours of induction the cells are harvested and assayed for phenotypic and
molecular parameters. Reduced GFP expression in siNA treated cells
(measured by flow cytometry) is taken as evidence for validation of the
target gene. Knockdown of target RNA in siNA treated cells can correlate
with reduced endogenous RNA and reduced GFP RNA to complete validation of
the target.

Example 16

Screening siNA Constructs for Improved Pharmacokinetics

[0716] In a non-limiting example, siNA constructs are screened in vivo for
improved pharmacokinetic properties compared to all RNA or unmodified
siNA constructs. Chemical modifications are introduced into the siNA
construct based on educated design parameters (e.g. introducing
2'-mofications, base modifications, backbone modifications, terminal cap
modifications, or covalently attached conjugates etc). The modified
construct in tested in an appropriate system (e.g human serum for
nuclease resistance, shown, or an animal model for PK/delivery
parameters). In parallel, the siNA construct is tested for RNAi activity,
for example in a cell culture system such as a luciferase reporter
assay). Lead siNA constructs are then identified which possess a
particular characteristic while maintaining RNAi activity, and can be
further modified and assayed once again. This same approach can be used
to identify siNA-conjugate molecules with improved pharmacokinetic
profiles, delivery, localized delivery, cellular uptake, and RNAi
activity.

Example 17

Indications

[0717] The siNA molecules of the invention can be used to treat a variety
of diseases and conditions through modulation of gene expression. Using
the methods described herein, chemically modified siNA molecules can be
designed to modulate the expression any number of target genes, including
but not limited to genes associated with cancer, metabolic diseases,
infectious diseases such as viral, bacterial or fungal infections,
neurologic diseases, musculoskeletal diseases, diseases of the immune
system, diseases associated with signaling pathways and cellular
messengers, and diseases associated with transport systems including
molecular pumps and channels.

[0720] The siNA molecule of the invention can also be used in a variety of
agricultural applications involving modulation of endogenous or exogenous
gene expression in plants using siNA, including use as insecticidal,
antiviral and anti-fungal agents or modulate plant traits such as oil and
starch profiles and stress resistance.

Example 18

Diagnostic Uses

[0721] The siNA molecules of the invention can be used in a variety of
diagnostic applications, such as in the identification of molecular
targets (e.g., RNA) in a variety of applications, for example, in
clinical, industrial, environmental, agricultural and/or research
settings. Such diagnostic use of siNA molecules involves utilizing
reconstituted RNAi systems, for example, using cellular lysates or
partially purified cellular lysates. siNA molecules of this invention can
be used as diagnostic tools to examine genetic drift and mutations within
diseased cells or to detect the presence of endogenous or exogenous, for
example viral, RNA in a cell. The close relationship between siNA
activity and the structure of the target RNA allows the detection of
mutations in any region of the molecule, which alters the base-pairing
and three-dimensional structure of the target RNA. By using multiple siNA
molecules described in this invention, one can map nucleotide changes,
which are important to RNA structure and function in vitro, as well as in
cells and tissues. Cleavage of target RNAs with siNA molecules can be
used to inhibit gene expression and define the role of specified gene
products in the progression of disease or infection. In this manner,
other genetic targets can be defined as important mediators of the
disease. These experiments will lead to better treatment of the disease
progression by affording the possibility of combination therapies (e.g.,
multiple siNA molecules targeted to different genes, siNA molecules
coupled with known small molecule inhibitors, or intermittent treatment
with combinations siNA molecules and/or other chemical or biological
molecules). Other in vitro uses of siNA molecules of this invention are
well known in the art, and include detection of the presence of mRNAs
associated with a disease, infection, or related condition. Such RNA is
detected by determining the presence of a cleavage product after
treatment with a siNA using standard methodologies, for example,
fluorescence resonance emission transfer (FRET).

[0722] In a specific example, siNA molecules that cleave only wild-type or
mutant forms of the target RNA are used for the assay. The first siNA
molecules (i.e., those that cleave only wild-type forms of target RNA)
are used to identify wild-type RNA present in the sample and the second
siNA molecules (i.e., those that cleave only mutant forms of target RNA)
are used to identify mutant RNA in the sample. As reaction controls,
synthetic substrates of both wild-type and mutant RNA are cleaved by both
siNA molecules to demonstrate the relative siNA efficiencies in the
reactions and the absence of cleavage of the "non-targeted" RNA species.
The cleavage products from the synthetic substrates also serve to
generate size markers for the analysis of wild-type and mutant RNAs in
the sample population. Thus, each analysis requires two siNA molecules,
two substrates and one unknown sample, which is combined into six
reactions. The presence of cleavage products is determined using an RNase
protection assay so that full-length and cleavage fragments of each RNA
can be analyzed in one lane of a polyacrylamide gel. It is not absolutely
required to quantify the results to gain insight into the expression of
mutant RNAs and putative risk of the desired phenotypic changes in target
cells. The expression of mRNA whose protein product is implicated in the
development of the phenotype (i.e., disease related or infection related)
is adequate to establish risk. If probes of comparable specific activity
are used for both transcripts, then a qualitative comparison of RNA
levels is adequate and decreases the cost of the initial diagnosis.
Higher mutant form to wild-type ratios are correlated with higher risk
whether RNA levels are compared qualitatively or quantitatively.

Example 19

Synthesis of siNA Conjugates

[0723] The introduction of conjugate moieties to siNA molecules of the
invention is accomplished either during solid phase synthesis using
phosphoramidite chemistry described above, or post-synthetically using,
for example, N-hydroxysuccinimide (NHS) ester coupling to an amino linker
present in the siNA. Typically, a conjugate introduced during solid phase
synthesis will be added to the 5'-end of a nucleic acid sequence as the
final coupling reaction in the synthesis cycle using the phosphoramidite
approach. Coupling conditions can be optimized for high yield coupling,
for example by modification of coupling times and reagent concentrations
to effectuate efficient coupling. As such, the 5'-end of the sense strand
of a siNA molecule is readily conjugated with a conjugate moiety having a
reactive phosphorus group available for coupling (e.g., a compound having
Formulae 1, 5, 8, 55, 56, 57, 60, 86, 92, 104, 110, 113, 115, 116, 117,
118, 120, or 122) using the phosphoramidite approach, providing a
5'-terminal conjugate (see for example FIG. 65).

[0724] Conjugate precursors having a reactive phosphorus group and a
protected hydroxyl group can be used to incorporate a conjugate moiety
anywhere in the siNA sequence, such as in the loop portion of a single
stranded hairpin siNA construct (see for example FIG. 66). For example,
using the phosphoramidite approach, a conjugate moiety comprising a
phosphoramidite and protected hydroxyl (e.g., a compound having Formulae
86, 92, 104, 113, 115, 116, 117, 118, 120, or 122 herein) is first
coupled at the desired position within the siNA sequence using solid
phase synthesis phosphoramidite coupling. Second, removal of the
protecting group (e.g., dimethoxytrityl) allows coupling of additional
nucleotides to the siNA sequence. This approach allows the conjugate
moiety to be positioned anywhere within the siNA molecule.

[0725] Conjugate derivatives can also be introduced to a siNA molecule
post synthetically. Post synthetic conjugation allows a conjugate moiety
to be introduced at any position within the siNA molecule where an
appropriate functional group is present (e.g., a C5 alkylamine linker
present on a nucleotide base or a 2'-alkylamine linker present on a
nucleotide sugar can provide a point of attachment for an NHS-conjugate
moiety). Generally, a reactive chemical group present in the siNA
molecule is unmasked following synthesis, thus allowing post-synthetic
coupling of the conjugate to occur. In a non-limiting example, an
protected amino linker containing nucleotide (e.g., TFA protected C5
propylamino thymidine) is introduced at a desired position of the siNA
during solid phase synthesis. Following cleavage and deprotection of the
siNA, the free amine is made available for NHS ester coupling of the
conjugate at the desired position within the siNA sequence, such as at
the 3'-end of the sense and/or antisense strands, the 3' and/or 5'-end of
the sense strand, or within the siNA sequence, such as in the loop
portion of a single stranded hairpin siNA sequence.

[0726] A conjugate moiety can be introduced at different locations within
a siNA molecule using both solid phase synthesis and post-synthetic
coupling approaches. For example, solid phase synthesis can be used to
introduce a conjugate moiety at the 5'-end of the siNA (e.g. sense
strand) and post-synthetic coupling can be used to introduce a conjugate
moiety at the 3'-end of the siNA (e.g. sense strand and/or antisense
strand). As such, a siNA sense strand having 3' and 5' end conjugates can
be synthesized (see for example FIG. 65). Conjugate moieties can also be
introduced in other combinations, such as at the 5'-end, 3'-end and/or
loop portions of a siNA molecule (see for example FIG. 66).

[0734] The Cholesterol, Phospholipid, and PEG conjugates were evaluated
for pharmakokinetic properties in mice compared to a non-conjugated siNA
construct having matched chemistry and sequence. This study was conducted
in female CD-1 mice approximately 26 g (6-7 weeks of age). Animals were
housed in groups of 3. Food and water were provided ad libitum.
Temperature and humidity were according to Pharmacology Testing Facility
performance standards (SOP's) which are in accordance with the 1996 Guide
for the Care and Use of Laboratory Animals (NRC). Animals were acclimated
to the facility for at least 3 days prior to experimentation.

[0735] Absorbance at 260 nm was used to determine the actual concentration
of the stock solution of pre-annealed HBV siNA. An appropriate amount of
HBV siNA was diluted in sterile veterinary grade normal saline (0.9%)
based on the average body weight of the mice. A small amount of the
antisense (Stab 7) strand was internally labeled with gamma 32P-ATP. The
32P-labeled stock was combined with excess sense strand (Stab 8) and
annealed. Annealing was confirmed prior to combination with unlabeled
drug. Each mouse received a subcutaneous bolus of 30 mg/kg (based on
duplex) and approximately 10 million cpm (specific activity of
approximately 15 cpm/ng).

[0736] Three animals per timepoint (1, 4, 8, 24, 72, 96 h) were euthanized
by CO2 inhalation followed immediately by exsanguination. Blood was
sampled from the heart and collected in heparinized tubes. After
exsanguination, animals were perfused with 10-15 mL of sterile veterinary
grade saline via the heart. Samples of liver were then collected and
frozen.

[0737] Tissue samples were homogenized in a digestion buffer prior to
compound quantitation. Quantitation of intact compound was determined by
scintillation counting followed by PAGE and phosphorimage analysis.
Results are shown in FIG. 43. As shown in the figure, the conjugated siNA
constructs shown vastly improved liver PK compared to the unconjugated
siNA construct.

Example 21

Cell Culture of siNA Conjugates (FIG. 68)

[0738] The Cholesterol conjugates and Phospholipid conjugated siNA
constructs described in Example 20 above were evaluated for cell culture
efficacy in a HBV cell culture system.

Transfection of HepG2 Cells with psHBV-1 and siNA

[0739] The human hepatocellular carcinoma cell line Hep G2 was grown in
Dulbecco's modified Eagle media supplemented with 10% fetal calf serum, 2
mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 25
mM Hepes, 100 units penicillin, and 100 μg/ml streptomycin. To
generate a replication competent cDNA, prior to transfection the HBV
genomic sequences are excised from the bacterial plasmid sequence
contained in the psHBV-1 vector. Other methods known in the art can be
used to generate a replication competent cDNA. This was done with an
EcoRI and Hind III restriction digest. Following completion of the
digest, a ligation was performed under dilute conditions (20 μg/ml) to
favor intermolecular ligation. The total ligation mixture was then
concentrated using Qiagen spin columns.

siNA Activity Screen and Dose Response Assay

[0740] Transfection of the human hepatocellular carcinoma cell line, Hep
G2, with replication-competent HBV DNA results in the expression of HBV
proteins and the production of virions. To test the efficacy of siNA
conjugates targeted against HBV RNA, the Cholesterol siNA conjugate and
Phospholipid siNA conjugate described in Example 12 were compared to a
non-conjugated control siNA (see FIG. 68). An inverted sequence duplex
was used as a negative control for the unconjugated siNA. Dose response
studies were performed in which HBV genomic DNA was transfected with HBV
genomic DNA with lipid at 12.5 ug/ml into Hep G2 cells. 24 hours after
transfection with HBV DNA, cell culture media was removed and siNA
duplexes were added to cells without lipid at 10 uM, 5, uM, 2.5 uM, 1 uM,
and 100 nm and the subsequent levels of secreted HBV surface antigen
(HBsAg) were analyzed by ELISA 72 hours post treatment (see FIG. 44). To
determine siNA activity, HbsAg levels were measured following
transfection with siNA. Immulon 4 (Dynax) microtiter wells were coated
overnight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay)
at 1 μg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5).
The wells were then washed 4× with PBST (PBS, 0.05% Tween® 20)
and blocked for 1 hr at 37° C. with PBST, 1% BSA. Following
washing as above, the wells were dried at 37° C. for 30 min.
Biotinylated goat ant-HBsAg (Accurate YVS1807) was diluted 1:1000 in PBST
and incubated in the wells for 1 hr. at 37° C. The wells were
washed 4× with PBST. Streptavidin/Alkaline Phosphatase Conjugate
(Pierce 21324) was diluted to 250 ng/ml in PBST, and incubated in the
wells for 1 hr. at 37° C. After washing as above, p-nitrophenyl
phosphate substrate (Pierce 37620) was added to the wells, which were
then incubated for 1 hour at 37° C. The optical density at 405 nm
was then determined. As shown in FIG. 68, the phospholipid and
cholesterol conjugates demonstrate marked dose dependent inhibition of
HBsAg expression compared to the unconjugated siNA construct when
delivered to cells without any transfection agent (lipid).

Example 22

Ex Vivo Stability of siNA Constructs

[0741] Chemically modified siNA constructs were designed and synthesized
in order to improve resistance to nucleases while maintaining silencing
in cell culture systems. Modified strands, designated Stab 4, Stab 5,
Stab 7, Stab 8, and Stab 11 (Table IV), were tested in three sets of
duplexes that demonstrated a range of stability and activity. These
duplexes contained differentially modified sense and antisense strands.
All modified sense strands contain terminal 5' and 3' inverted abasic
caps, while antisense strands possess a 3' terminal phosphorothioate
linkage. The results characterize the impact of chemical modifications on
nuclease resistance in ex vivo models of the environments sampled by
drugs.

[0742] Active siNAs were assessed for their resistance to degradation in
serum and liver extracts. Stability in blood will be a requirement for a
systemically administered siNA, and an anti-HBV or anti-HCV siNA would
require stability and activity in the hepatic intracellular environment.
Liver extracts potentially provide an extreme nuclease model where many
catabolic enzymes are present. Both mouse and human systems were
assessed.

[0743] Individual strands of siNA duplexes were internally labeled with
32P and incubated as single strands or as duplex siRNAs in human or mouse
serum and liver extracts. Representative data is shown in Table VI.
Throughout the course of the experiments, constant levels of ribonuclease
activity were verified. The extent and pattern of all-RNA siNA
degradation (3 minute time point) did not change following preincubation
of serum or liver extract at 37° C. for up to 24 hours.

[0744] The biological activity of siRNAs containing all-ribose residues
has been well established. The extreme instability (t 1/2=0.017 hours) of
these compounds in serum underscores the need for chemical modification
for use in systemic therapeutic applications. The Stab 4/5 duplex
modifications provide significant stability in human and mouse serum (t
1/2's=10-408 hours) and human liver extract (t 1/2's=28-43 hours). In
human serum the Stab 4 strand chemistry in the context of the Stab 4/5
duplex, possesses greater stability than the Stab 5 strand chemistry (t
1/2=408 vs. 39 hours). This result highlights the impact terminal
modifications have on stability. A fully-modified Stab 7/11 construct (no
ribonucleotides present) was generated from the Stab 4/5 constructs by
substituting the ribonucleotides in all purine positions with
deoxyribonucleotides. Another fully modified construct, Stab 7/8, was
generated by replacing all purine positions in the antisense strand with
2'-O-methyl nucleotides. This proved to be the most stable antisense
strand chemistry observed, with t 1/2=816 hours in human liver extract.

[0745] The dramatic stability of Stab 8 modifications was also observed
when non-duplexed single strands were incubated in human serum and liver
extract, as shown in Table VII. An approximate five-fold increase in
serum stability is seen for the double stranded constructs, compared to
that observed for the individual strands. In liver extract, the siNA
duplex provides even greater stability compared to the single strands.
For example, the Stab 5 chemistry is greater than 100-fold more stable in
the Stab 4/5 duplex relative to its stability alone.

[0746] Terminal modifications have a large impact on stability in human
serum, as can be seen from a comparison of sense verses antisense
stabilities in duplex form, and the Stab 4 and Stab 5 single-strand
stabilities. Therefore, a number of 3' antisense capping moieties on Stab
4/5 chemistry duplexes were assessed for their contribution to stability
in human serum. The structures of these modifications are shown in FIG.
22, and resultant half-lives are shown in Table VIII. A wide range of
different stabilities were observed, from half-lives as short as one hour
to greater than 770 hours. Thus, in the context of 2'-fluoro modified
pyrimidines, 3'-exonuclease becomes the primary mode of attack on
duplexes in human serum; a number of chemistries minimize this site of
attack. These results suggest that susceptibility to 3' exonucleases is a
major path to degradation in the serum.

Example 23

Activity of siNA Molecules Delivered Via Hydrodynamic Injection

[0747] An in vivo mouse model that utilizes hydrodynamic tail vein
injection of a replication competent HBV vector has been used to assess
the activity of chemically stabilized siRNA targeted to HBV RNA. The
hydrodynamic delivery of nucleic acids in the mouse has been described by
Liu et al., 1999, Gene Therapy, 6, 1258-1266, who showed that the vast
majority of the nucleic acid is delivered to the liver by this technique.
The use of the hydrodynamic technology to develop an HBV mouse model has
been described by Yang et al., 2002, PNAS, 99, 13825-13830. In the
vector-based model, HBV replicates in the liver for approximately 10
days, resulting in detectable levels of HBV RNA and antigens in the liver
and HBV DNA and antigens in the serum.

[0748] To assess the activity of chemically stabilized siNAs against HBV,
co-injection of the siNAs along with the HBV vector was done in mouse
strain C57BL/J6. The HBV vector used, pWTD, is a head-to-tail dimer of
the complete HBV genome (see for example Buckwold et al., 1996, J.
Virology, 70, 5845-5851). For a 20 gram mouse, a total injection of 1.6
ml containing 10 μg or 1 μg of pWTD and 100 μg of siNA duplex in
saline, was injected into the tail vein within 5 seconds. For a larger
mouse, the volume is scaled to maintain a level of 140% of the blood
volume of the mouse. The injection is done using a 3 cc syringe and 27
g1/2 needle. The animals were sacrificed at 72 hrs post-injection.
Animals were treated with siNA constructs and matched chemistry inverted
controls. Analysis of the HBV DNA (FIG. 80) and HBsAg (FIG. 81) levels in
serum was conducted by real-time PCR and ELISA respectively. The levels
of HBV RNA in the liver (FIG. 82) were analyzed by real-time RT-PCR. In a
separate experiment, analysis of HBV DNA levels in serum was carried out
at 5 days and 7 days (FIG. 83) after co-injection of siNA and the HBV
vector.

Example 24

Activity Screens Using Chemically Modified siNA

[0749] Two formats can be used to identify active chemically modified siNA
molecules against target nucleic acid molecules (e.g., RNA). One format
involves screening unmodified siNA constructs in an appropriate system
(e.g., cell culture or animal models) then applying chemical
modifications to the sequence of identified leads and rescreening the
modified constructs. Another format involves direct screening of
chemically modified constructs to identify chemically modified leads (see
for example the Stab 7/8 HCV screen shown in FIG. 86 and the Stab 7/8 HBV
screen shown FIG. 87, as described above). The latter approach can be
useful in identifying active constructs that are specific to various
combinations of chemical modifications (e.g., Stab 1-18 chemistries shown
in Table V herein). Additionally, different iterations of such chemical
modifications can be assessed using active chemically modified leads and
appropriate rules for selective active constructs given a particular
chemisty can be established using this approach. Non-limiting examples of
such activity screen are described below.

Activity Screen of Stab 7/8 Constructs Targeting Luciferase RNA

[0750] HeLa cells were co-transfected with pGF3 vector (250 ng/well),
renilla luciferase vector (10 ng/well) and siNA (0.5-25 nM) using 0.5 ul
lipofectamine-2000 per well. Twenty-four hours post-transfection, the
cells were assayed for luciferase activity using the Promega Dual
Luciferase Assay Kit per the manufacturer's instruction. siNA constructs
having high levels of activity were identified and tested in a dose
response assay with concentrations ranging from 0.5 to 25 nM. Results for
siNA constructs targeting sites 80, 237, and 1478 are shown in FIG. 84
and sites 1544 and 1607 are shown in FIG. 85. As shown in the Figures,
several active Stab 7/8 constructs were identified that demonstrate
potent dose related inhibition of luciferase expression.

[0751] The HBV HepG2 cell culture system described in Example 13 above was
utilized to evaluate the efficacy of various combinations of chemical
modifications (Table V) in the sense strand and antisense strand of siNA
molecules as compared to matched chemistry inverted controls. To
determine siNA activity, HbsAg levels were measured following
transfection with siNA. Immulon 4 (Dynax) microtiter wells were coated
overnight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay)
at 1 μg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5).
The wells were then washed 4× with PBST (PBS, 0.05% Tween® 20)
and blocked for 1 hr at 37° C. with PBST, 1% BSA. Following
washing as above, the wells were dried at 37° C. for 30 min.
Biotinylated goat ant-HBsAg (Accurate YVS1807) was diluted 1:1000 in PBST
and incubated in the wells for 1 hr. at 37° C. The wells were
washed 4× with PBST. Streptavidin/Alkaline Phosphatase Conjugate
(Pierce 21324) was diluted to 250 ng/ml in PBST, and incubated in the
wells for 1 hr. at 37° C. After washing as above, p-nitrophenyl
phosphate substrate (Pierce 37620) was added to the wells, which were
then incubated for 1 hour at 37° C. The optical density at 450 nm
was then determined. Results of the combination HBV siNA screen are shown
in FIGS. 88-90. As shown in the Figures, the various combinations of
differing sense and antisense chemistries (e.g., sense/antisense
constructs having Stab 7/8, 7/10, 7/11, 9/8, 9/10, 6/10, 16/8, 16/10,
18/8, 18/10, 4/8, 4/10, 7/5, 9/5, and 9/11 chemistry) result in active
siNA constructs.

[0752] All patents and publications mentioned in the specification are
indicative of the levels of skill of those skilled in the art to which
the invention pertains. All references cited in this disclosure are
incorporated by reference to the same extent as if each reference had
been incorporated by reference in its entirety individually.

[0753] One skilled in the art would readily appreciate that the present
invention is well adapted to carry out the objects and obtain the ends
and advantages mentioned, as well as those inherent therein. The methods
and compositions described herein as presently representative of
preferred embodiments are exemplary and are not intended as limitations
on the scope of the invention. Changes therein and other uses will occur
to those skilled in the art, which are encompassed within the spirit of
the invention, are defined by the scope of the claims.

[0754] It will be readily apparent to one skilled in the art that varying
substitutions and modifications can be made to the invention disclosed
herein without departing from the scope and spirit of the invention.
Thus, such additional embodiments are within the scope of the present
invention and the following claims. The present invention teaches one
skilled in the art to test various combinations and/or substitutions of
chemical modifications described herein toward generating nucleic acid
constructs with improved activity for mediating RNAi activity. Such
improved activity can comprise improved stability, improved
bioavailability, and/or improved activation of cellular responses
mediating RNAi. Therefore, the specific embodiments described herein are
not limiting and one skilled in the art can readily appreciate that
specific combinations of the modifications described herein can be tested
without undue experimentation toward identifying siNA molecules with
improved RNAi activity.

[0755] The invention illustratively described herein suitably can be
practiced in the absence of any element or elements, limitation or
limitations that are not specifically disclosed herein. Thus, for
example, in each instance herein any of the terms "comprising",
"consisting essentially of", and "consisting of" may be replaced with
either of the other two terms. The terms and expressions which have been
employed are used as terms of description and not of limitation, and
there is no intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are possible
within the scope of the invention claimed. Thus, it should be understood
that although the present invention has been specifically disclosed by
preferred embodiments, optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled in the
art, and that such modifications and variations are considered to be
within the scope of this invention as defined by the description and the
appended claims.

[0756] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of alternatives,
those skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group or other group.